Nucleoside phosphoramidates

ABSTRACT

Disclosed herein are nucleoside phosphoramidates and their use as agents for treating viral diseases. These compounds are inhibitors of RNA-dependent 5 RNA viral replication and are useful as inhibitors of HCV NS5B polymerase, as inhibitors of HCV replication and for treatment of hepatitis C infection in mammals.

PRIORITY CLAIM

The right of priority is claimed to U.S. Provisional Patent ApplicationNos. 61/179,923, filed May 20, 2009, and 61/319,513, filed Mar. 31,2010, the subject matter of which is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

Disclosed herein are nucleoside phosphoramidates and their use as agentsfor treating viral diseases. These compounds are inhibitors ofRNA-dependent RNA viral replication and are useful as inhibitors of HCVNS5B polymerase, as inhibitors of HCV replication and for treatment ofhepatitis C infection in mammals.

BACKGROUND

Hepatitis C virus (HCV) infection is a major health problem that leadsto chronic liver disease, such as cirrhosis and hepatocellularcarcinoma, in a substantial number of infected individuals, estimated tobe 2-15% of the world's population. There are an estimated 4.5 millioninfected people in the United States alone, according to the U.S. Centerfor Disease Control. According to the World Health Organization, thereare more than 200 million infected individuals worldwide, with at least3 to 4 million people being infected each year. Once infected, about 20%of people clear the virus, but the rest can harbor HCV the rest of theirlives. Ten to twenty percent of chronically infected individualseventually develop liver-destroying cirrhosis or cancer. The viraldisease is transmitted parenterally by contaminated blood and bloodproducts, contaminated needles, or sexually and vertically from infectedmothers or carrier mothers to their offspring. Current treatments forHCV infection, which are restricted to immunotherapy with recombinantinterferon-α alone or in combination with the nucleoside analogribavirin, are of limited clinical benefit. Moreover, there is noestablished vaccine for HCV. Consequently, there is an urgent need forimproved therapeutic agents that effectively combat chronic HCVinfection.

The HCV virion is an enveloped positive-strand RNA virus with a singleoligoribonucleotide genomic sequence of about 9600 bases which encodes apolyprotein of about 3,010 amino acids. The protein products of the HCVgene consist of the structural proteins C, E1, and E2, and thenon-structural proteins NS2, NS3, NS4A and NS4B, and NS5A and NS5B. Thenonstructural (NS) proteins are believed to provide the catalyticmachinery for viral replication. The NS3 protease releases NS5B, theRNA-dependent RNA polymerase from the polyprotein chain. HCV NS5Bpolymerase is required for the synthesis of a double-stranded RNA from asingle-stranded viral RNA that serves as a template in the replicationcycle of HCV. Therefore, NS5B polymerase is considered to be anessential component in the HCV replication complex (K. Ishi, et al,Heptology, 1999, 29: 1227-1235; V. Lohmann, et al., Virology, 1998, 249:108-118). Inhibition of HCV NS5B polymerase prevents formation of thedouble-stranded HCV RNA and therefore constitutes an attractive approachto the development of HCV-specific antiviral therapies.

HCV belongs to a much larger family of viruses that share many commonfeatures.

Flaviviridae Viruses

The Flaviviridae family of viruses comprises at least three distinctgenera: pestiviruses, which cause disease in cattle and pigs;flavivruses, which are the primary cause of diseases such as denguefever and yellow fever; and hepaciviruses, whose sole member is HCV. Theflavivirus genus includes more than 68 members separated into groups onthe basis of serological relatedness (Calisher et al., J. Gen. Virol,1993, 70,37-43). Clinical symptoms vary and include fever, encephalitisand hemorrhagic fever (Fields Virology, Editors: Fields, B. N., Knipe,D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia,Pa., 1996, Chapter 31, 931-959). Flaviviruses of global concern that areassociated with human disease include the Dengue Hemorrhagic Feverviruses (DHF), yellow fever virus, shock syndrome and Japaneseencephalitis virus (Halstead, S. B., Rev. Infect. Dis., 1984, 6,251-264; Halstead, S. B., Science, 239:476-481, 1988; Monath, T. P., NewEng. J. Med, 1988, 319, 64 1-643).

The pestivirus genus includes bovine viral diarrhea virus (BVDV),classical swine fever virus (CSFV, also called hog cholera virus) andborder disease virus (BDV) of sheep (Moennig, V. et al. Adv. Vir. Res.1992, 41, 53-98). Pestivirus infections of domesticated livestock(cattle, pigs and sheep) cause significant economic losses worldwide.BVDV causes mucosal disease in cattle and is of significant economicimportance to the livestock industry (Meyers, G. and Thiel, H. J.,Advances in Virus Research, 1996, 47, 53-118; Moennig V., et al, Adv.Vir. Res. 1992, 41, 53-98). Human pestiviruses have not been asextensively characterized as the animal pestiviruses. However,serological surveys indicate considerable pestivirus exposure in humans.

Pestiviruses and hepaciviruses are closely related virus groups withinthe Flaviviridae family. Other closely related viruses in this familyinclude the GB virus A, GB virus A-like agents, GB virus-B and GBvirus-C (also called hepatitis G virus, HGV). The hepacivirus group(hepatitis C virus; HCV) consists of a number of closely related butgenotypically distinguishable viruses that infect humans. There are atleast 6 HCV genotypes and more than 50 subtypes. Due to the similaritiesbetween pestiviruses and hepaciviruses, combined with the poor abilityof hepaciviruses to grow efficiently in cell culture, bovine viraldiarrhea virus (BVDV) is often used as a surrogate to study the HCVvirus.

The genetic organization of pestiviruses and hepaciviruses is verysimilar. These positive stranded RNA viruses possess a single large openreading frame (ORF) encoding all the viral proteins necessary for virusreplication. These proteins are expressed as a polyprotein that is co-and post-translationally processed by both cellular and virus-encodedproteinases to yield the mature viral proteins. The viral proteinsresponsible for the replication of the viral genome RNA are locatedwithin approximately the carboxy-terminal. Two-thirds of the ORF aretermed nonstructural (NS) proteins. The genetic organization andpolyprotein processing of the nonstructural protein portion of the ORFfor pestiviruses and hepaciviruses is very similar. For both thepestiviruses and hepaciviruses, the mature nonstructural (NS) proteins,in sequential order from the amino-terminus of the nonstructural proteincoding region to the carboxy-terminus of the ORF, consist of p7, NS2,NS3, NS4A, NS4B, NS5A, and NS5B.

The NS proteins of pestiviruses and hepaciviruses share sequence domainsthat are characteristic of specific protein functions. For example, theNS3 proteins of viruses in both groups possess amino acid sequencemotifs characteristic of serine proteinases and of helicases (Gorbalenyaet al., Nature, 1988, 333, 22; Bazan and Fletterick Virology, 1989, 171,637-639; Gorbalenya et al., Nucleic Acid Res., 1989, 17, 3889-3897).Similarly, the NS5B proteins of pestiviruses and hepaciviruses have themotifs characteristic of RNA-directed RNA polymerases (Koonin, E. V. andDolja, V. V., Crir. Rev. Biochem. Molec. Biol. 1993, 28, 375-430).

The actual roles and functions of the NS proteins of pestiviruses andhepaciviruses in the lifecycle of the viruses are directly analogous. Inboth cases, the NS3 serine proteinase is responsible for all proteolyticprocessing of polyprotein precursors downstream of its position in theORF (Wiskerchen and Collett, Virology, 1991, 184, 341-350;Bartenschlager et al., J. Virol. 1993, 67, 3835-3844; Eckart et al.Biochem. Biophys. Res. Comm. 1993, 192, 399-406; Grakoui et al., J.Virol. 1993, 67, 2832-2843; Grakoui et al., Proc. Natl. Acad. Sci. USA1993, 90, 10583-10587; Hijikata et al., J. Virol. 1993, 67, 4665-4675;Tome et al., J. Virol., 1993, 67, 4017-4026). The NS4A protein, in bothcases, acts as a cofactor with the NS3 serine protease (Bartenschlageret al., J. Virol. 1994, 68, 5045-5055; Failla et al., J. Virol. 1994,68, 3753-3760; Xu et al., J. Virol., 1997, 71:53 12-5322). The NS3protein of both viruses also functions as a helicase (Kim et al.,Biochem. Biophys. Res. Comm., 1995, 215, 160-166; Jin and Peterson,Arch. Biochem. Biophys., 1995, 323, 47-53; Warrener and Collett, J.Virol. 1995, 69, 1720-1726). Finally, the NS5B proteins of pestivirusesand hepaciviruses have the predicted RNA-directed RNA polymerasesactivity (Behrens et al., EMBO, 1996, 15, 12-22; Lechmann et al., J.Virol., 1997, 71, 8416-8428; Yuan et al., Biochem. Biophys. Res. Comm.1997, 232, 231-235; Hagedorn, PCT WO 97/12033; Zhong et al, J. Virol.,1998, 72, 9365-9369).

Currently, there are limited treatment options for individuals infectedwith hepatitis C virus. The current approved therapeutic option is theuse of immunotherapy with recombinant interferon-α alone or incombination with the nucleoside analog ribavirin. This therapy islimited in its clinical effectiveness and only 50% of treated patientsrespond to therapy. Therefore, there is significant need for moreeffective and novel therapies to address the unmet medical need posed byHCV infection.

A number of potential molecular targets for drug development of directacting antivirals as anti-HCV therapeutics have now been identifiedincluding, but not limited to, the NS2-NS3 autoprotease, the N3protease, the N3 helicase and the NS5B polymerase. The RNA-dependent RNApolymerase is absolutely essential for replication of thesingle-stranded, positive sense, RNA genome and this enzyme has elicitedsignificant interest among medicinal chemists.

Inhibitors of HCV NS5B as potential therapies for HCV infection havebeen reviewed: Tan, S.-L., et al., Nature Rev. Drug Discov., 2002, 1,867-881; Walker, M. P. et al., Exp. Opin. Investigational Drugs, 2003,12, 1269-1280; Ni, Z-J., et al., Current Opinion in Drug Discovery andDevelopment, 2004, 7, 446-459; Beaulieu, P. L., et al., Current Opinionin Investigational Drugs, 2004, 5, 838-850; Wu, J., et al., Current DrugTargets-Infectious Disorders, 2003, 3, 207-219; Griffith, R. C., et al,Annual Reports in Medicinal Chemistry, 2004, 39, 223-237; Carrol, S., etal., Infectious Disorders-Drug Targets, 2006, 6, 17-29. The potentialfor the emergence of resistant HCV strains and the need to identifyagents with broad genotype coverage supports the need for continuingefforts to identify novel and more effective nucleosides as HCV NS5Binhibitors.

Nucleoside inhibitors of NS5B polymerase can act either as a non-naturalsubstrate that results in chain termination or as a competitiveinhibitor which competes with nucleotide binding to the polymerase. Tofunction as a chain terminator the nucleoside analog must be taken up bythe cell and converted in vivo to a triphosphate to compete for thepolymerase nucleotide binding site. This conversion to the triphosphateis commonly mediated by cellular kinases which imparts additionalstructural requirements on a potential nucleoside polymerase inhibitor.Unfortunately, this limits the direct evaluation of nucleosides asinhibitors of HCV replication to cell-based assays capable of in situphosphorylation.

In some cases, the biological activity of a nucleoside is hampered byits poor substrate characteristics for one or more of the kinases neededto convert it to the active triphosphate form. Formation of themonophosphate by a nucleoside kinase is generally viewed as the ratelimiting step of the three phosphorylation events. To circumvent theneed for the initial phosphorylation step in the metabolism of anucleoside to the active triphosphate analog, the preparation of stablephosphate prodrugs has been reported. Nucleoside phosphoramidateprodrugs have been shown to be precursors of the active nucleosidetriphosphate and to inhibit viral replication when administered to viralinfected whole cells (McGuigan, C., et al., J. Med. Chem., 1996, 39,1748-1753; Valette, G., et al., J. Med. Chem., 1996, 39, 1981-1990;Balzarini, J., et al., Proc. National Acad Sci USA, 1996, 93, 7295-7299;Siddiqui, A. Q., et al., J. Med. Chem., 1999, 42, 4122-4128; Eisenberg,E. J., et al., Nucleosides, Nucleotides and Nucleic Acids, 2001, 20,1091-1098; Lee, W. A., et al., Antimicrobial Agents and Chemotherapy,2005, 49, 1898); US 2006/0241064; and WO 2007/095269.

Also limiting the utility of nucleosides as viable therapeutic agents istheir sometimes poor physicochemical and pharmacokinetic properties.These poor properties can limit the intestinal absorption of an agentand limit uptake into the target tissue or cell. To improve on theirproperties prodrugs of nucleosides have been employed. It has beendemonstrated that preparation of nucleoside phosphoramidates improvesthe systemic absorption of a nucleoside and furthermore, thephosphoramidate moiety of these “pronucleotides” is masked with neutrallipophilic groups to obtain a suitable partition coefficient to optimizeuptake and transport into the cell dramatically enhancing theintracellular concentration of the nucleoside monophosphate analogrelative to administering the parent nucleoside alone. Enzyme-mediatedhydrolysis of the phosphate ester moiety produces a nucleosidemonophosphate wherein the rate limiting initial phosphorylation isunnecessary. To this end, U.S. patent application Ser. No. 12/053,015,which corresponds to WO 2008/121634 and US 2010/0016251, discloses anumber of phosphoramidate nucleoside prodrugs, many of which showactivity in an HCV assay. Several compounds disclosed in US 2010/0016251were tested as a potential clinical candidate for approval by the FDA.

SUMMARY OF THE INVENTION

Disclosed herein is a compound represented by formula 4 and itsrespective phosphorus-based diastereomers represented by formulasS_(P)-4 and R_(P)-4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. High resolution XRD diffractogram of 4.

FIG. 2. High resolution XRD diffractogram of R_(P)-4.

FIG. 3. High resolution XRD diffractogram of S_(P)-4 (Form 1).

FIG. 4. High resolution XRD diffractogram of S_(P)-4 (Form 1).

FIG. 5. High resolution XRD diffractogram of S_(P)-4.CH₂Cl₂ (Form 2).

FIG. 6. High resolution XRD diffractogram of S_(P)-4.CHCl₃ (Form 3).

FIG. 7. High resolution XRD diffractogram of S_(P)-4 (Form 4).

FIG. 8. High resolution XRD diffractogram of S_(P)-4 (Form 5).

FIG. 9. High resolution XRD diffractogram of S_(P)-4 (amorphous).

FIG. 10. X-Ray Crystal Structure for S_(P)-4 (Form 1).

FIG. 11. X-Ray Crystal (Isotropic) Structure for S_(P)-4-CH₂Cl₂ (Form2).

FIG. 12. X-Ray Crystal (Anisotropic) Structure for S_(P)-4.CH₂Cl₂ (Form2).

FIG. 13. X-Ray Crystal Structure for S_(P)-4.CHCl₃ (Form 3).

FIG. 14. FT-IR spectrum of 4.

FIG. 15. FT-IR spectrum of R_(P)-4.

FIG. 16. FT-IR spectrum of S_(P)-4

FIG. 17. TGA and DSC analysis of 4.

FIG. 18. TGA and DSC analysis of R_(P)-4.

FIG. 19. TGA and DSC analysis of S_(P)-4.

FIG. 20A. X-Ray Crystal Structure for 8 (S_(P)-isomer) (molecule no. 1of the asymmetric unit).

FIG. 20B. X-Ray Crystal Structure for 8 (S_(P)-isomer) (molecule no. 2of the asymmetric unit).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The phrase “a” or “an” entity as used herein refers to one or more ofthat entity; for example, a compound refers to one or more compounds orat least one compound. As such, the terms “a” (or “an”), “one or more”,and “at least one” can be used interchangeably herein.

The terms “optional” or “optionally” as used herein means that asubsequently described event or circumstance may but need not occur, andthat the description includes instances where the event or circumstanceoccurs and instances in which it does not. For example, “optional bond”means that the bond may or may not be present, and that the descriptionincludes single, double, or triple bonds.

The term “P*” means that the phosphorus atom is chiral and that it has acorresponding Cahn-Ingold-Prelog designation of “R” or “S” which havetheir accepted plain meanings.

The term “purified,” as described herein, refers to the purity of agiven compound. For example, a compound is “purified” when the givencompound is a major component of the composition, i.e., at least 50% w/wpure. Thus, “purified” embraces at least 50% w/w purity, at least 60%w/w purity, at least 70% purity, at least 80% purity, at least 85%purity, at least 90% purity, at least 92% purity, at least 94% purity,at least 96% purity, at least 97% purity, at least 98% purity, at least99% purity, at least 99.5% purity, and at least 99.9% purity, wherein“substantially pure” embraces at least 97% purity, at least 98% purity,at least 99% purity, at least 99.5% purity, and at least 99.9% purity.

The term “metabolite,” as described herein, refers to a compoundproduced in vivo after administration to a subject in need thereof.

The term “about” (also represented by ˜) means that the recitednumerical value is part of a range that varies within standardexperimental error.

The expression “substantially as shown in . . . ” a specified XRPDpattern means that the peak positions shown in the XRPD pattern aresubstantially the same, within visual inspection or resort to selectedpeak listings (±0.2° 20). One of ordinary skill understands that theintensities can vary depending on the sample.

The term “substantially anhydrous” means that a substance contains atmost 10% by weight of water, preferably at most 1% by weight of water,more preferably at most 0.5% by weight of water, and most preferably atmost 0.1% by weight of water.

A solvent or anti-solvent (as used in reactions, crystallization, etc.or lattice and/or adsorbed solvents) includes at least one of a C₁ to C₈alcohol, a C₂ to C₈ ether, a C₃ to C₇ ketone, a C₃ to C₇ ester, a C₁ toC₂ chlorocarbon, a C₂ to C₇ nitrile, a miscellaneous solvent, a C₅ toC₁₂ saturated hydrocarbon, and a C₆ to C₁₂ aromatic hydrocarbon.

The C₁ to C₈ alcohol refers to a straight/branched and/or cyclic/acyclicalcohol having such number of carbons. The C₁ to C₈ alcohol includes,but is not limited to, methanol, ethanol, n-propanol, isopropanol,isobutanol, hexanol, and cyclohexanol.

The C₂ to C₈ ether refers to a straight/branched and/or cyclic/acyclicether having such number of carbons. The C₂ to C₈ ether includes, but isnot limited to, dimethyl ether, diethyl ether, di-isopropyl ether,di-n-butyl ether, methyl-t-butyl ether (MTBE), tetrahydrofuran, anddioxane

The C₃ to C₇ ketone refers to a straight/branched and/or cyclic/acyclicketone having such number of carbons. The C₃ to C₇ ketone includes, butis not limited to, acetone, methyl ethyl ketone, propanone, butanone,methyl isobutyl ketone, methyl butyl ketone, and cyclohexanone.

The C₃ to C₇ ester refers to a straight/branched and/or cyclic/acyclicester having such number of carbons. The C₃ to C₇ ester includes, but isnot limited to, ethyl acetate, propyl acetate, n-butyl acetate, etc.

The C₁ to C₂ chlorocarbon refers to a chlorocarbon having such number ofcarbons. The C₁ to C₂ chlorocarbon includes, but is not limited to,chloroform, methylene chloride (DCM), carbon tetrachloride,1,2-dichloroethane, and tetrachloroethane.

A C₂ to C₇ nitrile refers to a nitrile have such number of carbons. TheC₂ to C₇ nitrile includes, but is not limited to, acetonitrile,propionitrile, etc.

A miscellaneous solvent refers to a solvent commonly employed in organicchemistry, which includes, but is not limited to, diethylene glycol,diglyme (diethylene glycol dimethyl ether), 1,2-dimethoxy-ethane,dimethylformamide, dimethylsulfoxide, ethylene glycol, glycerin,hexamethylphsphoramide, hexamethylphosphorous triame,N-methyl-2-pyrrolidinone, nitromethane, pyridine, triethyl amine, andacetic acid.

The term C₅ to C₁₂ saturated hydrocarbon refers to a straight/branchedand/or cyclic/acyclic hydrocarbon. The C₅ to C₁₂ saturated hydrocarbonincludes, but is not limited to, n-pentane, petroleum ether (ligroine),n-hexane, n-heptane, cyclohexane, and cycloheptane.

The term C₆ to C₁₂ aromatic refers to substituted and unsubstitutedhydrocarbons having a phenyl group as their backbone. Preferredhydrocarbons include benzene, xylene, toluene, chlorobenzene, o-xylene,m-xylene, p-xylene, xylenes, with toluene being more preferred.

The term “halo” or “halogen” as used herein, includes chloro, bromo,iodo and fluoro.

The term “blocking group” refers to a chemical group which exhibits thefollowing characteristics. The “group” is derived from a “protectingcompound.” Groups that are selective for primary hydroxyls oversecondary hydroxyls that can be put on under conditions consistent withthe stability of the phosphoramidate (pH 2-8) and impart on theresulting product substantially different physical properties allowingfor an easier separation of the 3′-phosphoramidate-5′-new group productfrom the unreacted desired compound. The group must react selectively ingood yield to give a protected substrate that is stable to the projectedreactions (see Protective Groups in Organic Synthesis, 3^(nd) ed. T. W.Greene and P. G. M. Wuts, John Wiley & Sons, New York, N.Y., 1999).Examples of groups include, but are not limited to: benzoyl, acetyl,phenyl-substituted benzoyl, tetrahydropyranyl, trityl, DMT(4,4′-dimethoxytrityl), MMT (4-monomethoxytrityl), trimethoxytrityl,pixyl(9-phenylxanthen-9-yl) group, thiopixyl(9-phenylthioxanthen-9-yl)or 9-(p-methoxyphenyl)xanthine-9-yl (MOX), etc.; C(O)-alkyl, C(O)Ph,C(O)aryl, CH₂O-alkyl, CH₂O-aryl, SO₂-alkyl, SO₂-aryl,tert-butyldimethylsilyl, tert-butyldiphenylsilyl. Acetals, such as MOMor THP and the like are considered possible groups. Fluorinatedcompounds are also contemplated in so far that they can be attached tothe compound and can be selectively removed by passing through afluorous solid phase extraction media (FluoroFlash®). A specific exampleincludes a fluorinated trityl analog, trityl analog1-[4-(1H,1H,2H,2H-perfluorodecyl)phenyl)-1,1-diphenylmethanol. Otherfluorinated analogs of trityl, BOC, FMOC, CBz, etc. are alsocontemplated. Sulfonyl chlorides like p-toluenesulfonyl chloride canreact selectively on the 5′ position. Esters could be formed selectivelysuch as acetates and benzoates. Dicarboxylic anhydrides such as succinicanhydride and its derivatives can be used to generate an ester linkagewith a free carboxylic acid, such examples include, but are not limitedto oxalyl, malonyl, succinyl, glutaryl, adipyl, pimelyl, superyl,azelayl, sebacyl, phthalyl, isophthalyl, terephthalyl, etc. The freecarboxylic acid increases the polarity dramatically and can also be usedas a handle to extract the reaction product into mildy basic aqueousphases such as sodium bicarbonate solutions. The phosphoramidate groupis relatively stable in acidic media, so groups requiring acidicreaction conditions, such as, tetrahydropyranyl, could also be used.

The term “protecting group” which is derived from a “protectingcompound,” has its plain and ordinary meaning, i.e., at least oneprotecting or blocking group is bound to at least one functional group(e.g., —OH, —NH₂, etc.) that allows chemical modification of at leastone other functional group. Examples of protecting groups, include, butare not limited to, benzoyl, acetyl, phenyl-substituted benzoyl,tetrahydropyranyl, trityl, DMT (4,4′-dimethoxytrityl), MMT(4-monomethoxytrityl), trimethoxytrityl, pixyl (9-phenylxanthen-9-yl)group, thiopixyl (9-phenylthioxanthen-9-yl) or9-(p-methoxyphenyl)xanthine-9-yl (MOX), etc.; C(O)-alkyl, C(O)Ph,C(O)aryl, C(O)O(lower alkyl), C(O)O(lower alkylene)aryl (e.g.,—C(O)OCH₂Ph), C(O)Oaryl, CH₂O-alkyl, CH₂O-aryl, SO₂-alkyl, SO₂-aryl, aprotecting group comprising at least one silicon atom, such as,tert-butyldimethylsilyl, tert-butyldiphenylsilyl, Si(loweralkyl)₂OSi(lower alkyl)₂OH (such as, —Si(^(i)Pr)₂OSi(^(i)Pr)₂OH.

The term “protecting compound,” as used herein and unless otherwisedefined, refers to a compound that contains a “protecting group” andthat is capable of reacting with a compound that contains functionalgroups that are capable of being protected.

The term “leaving group”, as used herein, has the same meaning to theskilled artisan (Advanced Organic Chemistry: reactions, mechanisms andstructure—Fourth Edition by Jerry March, John Wiley and Sons Ed.; 1992pages 351-357) and represents a group which is part of and attached to asubstrate molecule; in a reaction where the substrate molecule undergoesa displacement reaction (with for example a nucleophile), the leavinggroup is then displaced. Examples of leaving groups include, but are notlimited to: halogen (F, Cl, Br, and I), preferably Cl, Br, or I;tosylate, mesylate, triflate, acetate, camphorsulfonate, aryloxide, andaryloxide substituted with at least one electron withdrawing group(e.g., p-nitrophenoxide, 2-chlorophenoxide, 4-chlorophenoxide,2,4-dinitrophenoxide, pentafluorophenoxide, etc.), etc. The term“electron withdrawing group” is accorded its plain meaning here.Examples of electron withdrawing groups include, but are not limited to,a halogen, —NO2, —C(O)(lower alkyl), —C(O)(aryl), —C(O)O(lower alkyl),—C(O)O(aryl), etc.

The term “basic reagent”, as used herein, means a compound that iscapable of deprotonating a hydroxyl group. Examples of basic reagentsinclude, but are not limited to, a (lower alk)oxide ((lower alkyl)OM) incombination with an alcoholic solvent, where (lower alk)oxides include,but are not limited to, MeO⁻, EtO⁻, ^(n)PrO⁻, ^(i)PrO⁻, ^(t)BuO⁻,^(i)AmO-(iso-amyloxide), etc., and where M is an alkali metal cation,such as Li⁺, Na⁺, K⁺, etc. Alcoholic solvents include (lower alkyl)OH,such as, for example, MeOH, EtOH, ^(n)PrOH, ^(i)PrOH, ^(t)BuOH,^(i)AmOH, etc. Non-alkoxy bases can also be used such as sodium hydride,sodium hexamethyldisilazane, lithium hexamethyldisilazane, lithiumdiisopropylamide, calcium hydride, sodium carbonate, potassiumcarbonate, cesium carbonate, DBU, DBN, Grignard reagents, such as (loweralkyl)Mg(halogen), which include but are not limited to MeMgCl, MeMgBr,^(t)BuMgCl, ^(t)BuMgBr, etc.

The term “base” embraces the term “basic reagent” and is meant to be acompound that is capable of deprotonating a proton containing compound,i.e., a Bronsted base. In addition to the examples recited above,further examples of a base include, but are not limited to pyridine,collidine, 2,6-(loweralkyl)-pyridine, dimethyl-aniline, imidazole,N-methyl-imidazole, pyrazole, N-methyl-pyrazole, triethylamine,di-isopropylethylamine, etc.

The term “electron withdrawing group” is accorded its plain meaningExamples of electron withdrawing groups include, but are not limited to,a halogen (F, Cl, Br, or I), —NO₂, —C(O)(lower alkyl), —C(O)(aryl),—C(O)O(lower alkyl), —C(O)O(aryl), etc.

The term “co-crystallates” include co-crystallates of 4, R_(P)-4, orS_(P)-4 in combination with salts, which embraces pharmaceuticallyacceptable salts.

The term “salts,” as described herein, refers to a compound comprising acation and an anion, which can produced by the protonation of aproton-accepting moiety and/or deprotonation of a proton-donatingmoiety. It should be noted that protonation of the proton-acceptingmoiety results in the formation of a cationic species in which thecharge is balanced by the presence of a physiological anion, whereasdeprotonation of the proton-donating moiety results in the formation ofan anionic species in which the charge is balanced by the presence of aphysiological cation.

The phrase “pharmaceutically acceptable salt” means a salt that ispharmaceutically acceptable. Examples of pharmaceutically acceptablesalts include, but are not limited to: (1) acid addition salts, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or formedwith organic acids such as glycolic acid, pyruvic acid, lactic acid,malonic acid, malic acid, maleic acid, fumaric acid, tartaric acid,citric acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, lauryl sulfuric acid,gluconic acid, glutamic acid, salicylic acid, muconic acid, and the likeor (2) basic addition salts formed with the conjugate bases of any ofthe inorganic acids listed above, wherein the conjugate bases comprise acationic component selected from among Na⁺, K⁺, Mg²⁺, Ca²⁺,NH_(g)R′″_(4-g) ⁺, in which R′″ is a C₁₋₃ alkyl and g is a numberselected from among 0, 1, 2, 3, or 4. It should be understood that allreferences to pharmaceutically acceptable salts include solvent additionforms (solvates) or crystal forms (polymorphs) as defined herein, of thesame acid addition salt.

The term “alkyl” refers to an unbranched or branched chain, saturated,monovalent hydrocarbon residue containing 1 to 30 carbon atoms. The term“C_(1-M) alkyl” refers to an alkyl comprising 1 to M carbon atoms, whereM is an integer having the following values: 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30. The term “C₁₋₄ alkyl” refers to an alkyl containing 1 to 4carbon atoms. The term “lower alkyl” denotes a straight or branchedchain hydrocarbon residue comprising 1 to 6 carbon atoms. “C₁₋₂₀ alkyl”as used herein refers to an alkyl comprising 1 to 20 carbon atoms.“C₁₋₁₀ alkyl” as used herein refers to an alkyl comprising 1 to 10carbons. Examples of alkyl groups include, but are not limited to, loweralkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl,t-butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. Theterm (ar)alkyl or (heteroaryl)alkyl indicate the alkyl group isoptionally substituted by an aryl or a heteroaryl group respectively.

The term “alkenyl” refers to an unsubstituted hydrocarbon chain radicalhaving from 2 to 10 carbon atoms having one or two olefinic doublebonds, preferably one olefinic double bond. The term “C_(2-N) alkenyl”refers to an alkenyl comprising 2 to N carbon atoms, where N is aninteger having the following values: 3, 4, 5, 6, 7, 8, 9, or 10. Theterm “C₂₋₁₀ alkenyl” refers to an alkenyl comprising 2 to 10 carbonatoms. The term “C₂₋₄ alkenyl” refers to an alkenyl comprising 2 to 4carbon atoms. Examples include, but are not limited to, vinyl,1-propenyl, 2-propenyl (allyl) or 2-butenyl (crotyl).

The term “aryl,” as used herein, and unless otherwise specified, refersto substituted or unsubstituted phenyl (Ph), biphenyl, or naphthyl,preferably the term aryl refers to substituted or unsubstituted phenyl.The aryl group can be substituted with one or more moieties selectedfrom among hydroxyl, F, Cl, Br, I, amino, alkylamino, arylamino, alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, and phosphonate, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin T. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” 3rd ed., John Wiley & Sons, 1999.

The term “aryloxide,” as used herein, and unless otherwise specified,refers to substituted or unsubstituted phenoxide (PhO—),p-phenyl-phenoxide (p-Ph-PhO—), or naphthoxide, preferably the termaryloxide refers to substituted or unsubstituted phenoxide. Thearyloxide group can be substituted with one or more moieties selectedfrom among hydroxyl, F, Cl, Br, I, —C(O)(lower alkyl), —C(O)O(loweralkyl), amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate,either unprotected, or protected as necessary, as known to those skilledin the art, for example, as taught in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis,” 3rd ed., John Wiley & Sons,1999.

The term “preparation” or “dosage form” is intended to include bothsolid and liquid formulations of the active compound and one skilled inthe art will appreciate that an active ingredient can exist in differentpreparations depending on the desired dose and pharmacokineticparameters.

The term “excipient” as used herein refers to a compound that is used toprepare a pharmaceutical composition, and is generally safe, non-toxicand neither biologically nor otherwise undesirable, and includesexcipients that are acceptable for veterinary use as well as humanpharmaceutical use.

The term “crystalline” refers to a situation where a solid sample ofeither S_(P)-4 or R_(P)-4 has crystalline characteristics whendetermined by X-ray powder diffraction or a single crystal X-raytechnique.

The term “crystal-like” refers to a situation where a solid sample ofeither S_(P)-4 or R_(P)-4 has crystalline characteristics whendetermined by one means, e.g., visually or by optical or polarizingmicroscopy, but does not have crystalline characteristics whendetermined by another means, e.g., x-ray powder diffraction. Methods ofvisually determining the crystallinity of a solid sample by visual or byoptical or by polarizing microscopy are disclosed in USP <695> and<776>, both of which are incorporated by reference. A solid sample ofeither S_(P)-4 or R_(P)-4 that is “crystal-like” may be crystallineunder certain conditions but may become non-crystalline when subjectedto other conditions.

The term “amorphous” refers to a situation where a solid sample ofeither S_(P)-4 or R_(P)-4 is neither crystalline nor crystal-like.

EMBODIMENTS

A first embodiment is directed to a compound represented by formula 4:

wherein P* represents a chiral phosphorus atom. Due to the chiralphosphorus atom, the compound represented by formula 4 comprises twodiastereomers designated as R_(P)-4 and S_(P)-4. The compoundrepresented by formula 4 can also be part of a solvate, a hydrate, or amixed solvate/hydrate. The solvate is designated as 4.nS, while thehydrate is designated as 4.mH₂O, where S is a lattice solvent, n variesby an integer or non-integer amount from about 0 to about 3 and m variesby an integer or non-integer amount from about 0 to about 5. Finally,the compound represented by formula 4 might not exist as a solvate orhydrate, but have a certain advantageous amount of adsorbed solvent (S)or water. In which case, the amount of S or water can vary from about 0wt. % to about 10 wt. % based on the weight of the compound representedby formula 4. The compound represented by formula 4 and its solvates andhydrates thereof is crystalline, crystal-like, or amorphous.

A second embodiment is directed to a compound represented by formulaR_(P)-4:

The compound represented by formula R_(P)-4 can also be part of asolvate, a hydrate, or a mixed solvate/hydrate. The solvate isdesignated as R_(P)-4.nS, while the hydrate is designated asS_(P)-4.mH₂O, where S is a lattice solvent, n varies by an integer ornon-integer amount from about 0 to about 3 and m varies by an integer ornon-integer amount from about 0 to about 5. Finally, the compoundrepresented by formula R_(P)-4 might not exist as a solvate, hydrate, ormixed solvate/hydrate, but have a certain advantageous amount ofadsorbed solvent (S), water, or both S and water. In which case, theamount of S or water can vary from about 0 wt. % to about 10 wt. % basedon the weight of the compound represented by formula R_(P)-4. Thecompound represented by formula R_(P)-4 and its solvates and hydratesthereof is crystalline, crystal-like, or amorphous.

A first aspect of the second embodiment is directed to crystallineR_(P)-4.

A second aspect of the second embodiment is directed to crystallineR_(P)-4 having XRPD 2θ-reflections (°) at about: 6.6, 7.1, 9.0, 11.6,17.9, 20.7, 24.1, 24.4, and 26.2.

A third aspect of the second embodiment is directed to a crystallineR_(P)-4 having XRPD 2θ-reflections (°) at about: 6.6, 7.1, 9.0, 11.0,11.6, 12.0, 16.0, 17.9, 19.6, 20.7, 21.0, 21.7, 21.9, 22.2, 23.1, 24.1,24.4, 26.1, 27.3, 27.7, and 28.2.

A fourth aspect of the second embodiment is directed to crystallineR_(P)-4 having an XRPD diffraction pattern substantially as that shownin FIG. 2.

A fifth aspect of the second embodiment is directed to R_(P)-4 havingthe following FT-IR peaks (cm⁻¹): 1742, 1713, 1679, 1460, 1377, 1259,1157, and 1079.

A sixth aspect of the second embodiment is directed to R_(P)-4 having anFT-IR spectrum substantially as that shown in FIG. 15.

A seventh aspect of the second embodiment is directed to substantiallypure R_(P)-4.

An eighth aspect of the second embodiment is directed to substantiallypure crystalline R_(P)-4.

A ninth aspect of the second embodiment is directed to substantiallypure amorphous R_(P)-4.

A third embodiment is directed to a compound represented by formulaS_(P)-4:

The compound represented by formula S_(P)-4 can also be part of asolvate, a hydrate, or a mixed solvate/hydrate. The solvate isdesignated as S_(P)-4.nS, while the hydrate is designated asS_(P)-4.mH₂O, where S is a lattice solvent, n varies in an integer ornon-integer amount from about 0 to about 3 and m varies in an integer ornon-integer amount from about 0 to about 5. Finally, the compoundrepresented by formula S_(P)-4 might not exist as a solvate or hydrate,but have a certain advantageous amount of adsorbed solvent (S) or water.In which case, the amount of S or water can vary from about 0 wt. % toabout 10 wt. % based on the weight of the compound represented byformula S_(P)-4. The compound represented by formula S_(P)-4 and itssolvates and hydrates thereof is crystalline, crystal-like, oramorphous.

A first aspect of the third embodiment is directed to crystallineS_(P)-4.

A second aspect of the third embodiment is directed to a monocliniccrystalline S_(P)-4, preferably having the following unit cellparameters a˜12.88 Å, b˜6.17 Å, c˜17.73 Å, and β˜92.05°.

A third aspect of the third embodiment is directed to a monocliniccrystalline S_(P)-4, preferably having the following unit cellparameters a˜20.09 Å, b˜6.10 Å, c˜23.01 Å, and β˜112.29°.

A fourth aspect of the third embodiment is directed to a monocliniccrystalline S_(P)-4, preferably having the following unit cellparameters a˜12.83 Å, b˜6.15 Å, c˜17.63 Å, and β˜91.75°.

A fifth aspect of the third embodiment is directed to a monocliniccrystalline S_(P)-4, preferably having the following unit cellparameters a˜12.93 Å, b˜6.18 Å, c˜18.01 Å, and β˜96.40°.

A sixth aspect of the third embodiment is directed to a crystallineS_(P)-4 having XRPD 2θ-reflections (°) at about: 5.2, 7.5, 9.6, 16.7,18.3, 22.2.

A seventh aspect of the third embodiment is directed to a crystallineS_(P)-4 having XRPD 2θ-reflections (°) at about: 5.0, 7.3, 9.4, and18.1.

An eighth aspect of the third embodiment is directed to a crystallineS_(P)-4 having XRPD 2θ-reflections (°) at about: 4.9, 6.9, 9.8, 19.8,20.6, 24.7, and 26.1.

A ninth aspect of the third embodiment is directed to a crystallineS_(P)-4 having XRPD 2θ-reflections (°) at about: 6.9, 9.8, 19.7, 20.6,and 24.6.

A ninth aspect of the third embodiment is directed to a crystallineS_(P)-4 having XRPD 2θ-reflections (°) at about: 5.0, 6.8, 19.9, 20.6,20.9, and 24.9.

A tenth aspect of the third embodiment is directed to a crystallineS_(P)-4 having XRPD 2θ-reflections (°) at about: 5.2, 6.6, 7.1, 15.7,19.1, and 25.0.

An eleventh aspect of the third embodiment is directed to crystallineS_(P)-4 having an XRPD diffraction pattern substantially as that shownin any one of FIG. 3, FIG. 4, FIG. 5, FIG. 6, FIG. 7, and FIG. 8.

A twelfth aspect of the third embodiment is directed to S_(P)-4 havingthe following FT-IR peaks (cm⁻¹) at about: 1743, 1713, 1688, 1454, 1378,1208, and 1082.

A thirteenth aspect of the third embodiment is directed to S_(P)-4having an FT-IR spectrum substantially as that shown in FIG. 7.

A fourteenth aspect of the third embodiment is directed to substantiallypure S_(P)-4.

A fifteenth aspect of the third embodiment is directed to substantiallypure crystalline S_(P)-4.

A sixteenth aspect of the third embodiment is directed to substantiallypure amorphous S_(P)-4.

Dosage, Administration, and Use

A fourth embodiment is directed to a composition for the treatmentand/or prophylaxis of any of the viral agents using any of compounds 4,R_(P)-4, or S_(P)-4. Possible viral agents include, but are not limitedto: hepatitis C virus, hepatitis B virus, Hepatitis A virus, West Nilevirus, yellow fever virus, dengue virus, rhinovirus, polio virus, bovineviral diarrhea virus, Japanese encephalitis virus, or those virusesbelonging to the groups of Pestiviruses, hepaciviruses, or flavaviruses.

An aspect of this embodiment is directed to a composition for thetreatment of any of the viral agents disclosed herein said compositioncomprising a pharmaceutically acceptable medium selected from among anexcipient, carrier, diluent, and equivalent medium and any of compounds4, R_(P)-4, or S_(P)-4, that is intended to include its hydrates,solvates, and any crystalline forms of any of compounds 4, R_(P)-4, orS_(P)-4 or its hydrates and solvates thereof.

The compounds 4, R_(P)-4, or S_(P)-4 may be independently formulated ina wide variety of oral administration dosage forms and carriers. Oraladministration can be in the form of tablets, coated tablets, hard andsoft gelatin capsules, solutions, emulsions, syrups, or suspensions. Thecompounds 4, R_(P)-4, or S_(P)-4 are efficacious when administered bysuppository administration, among other routes of administration. Themost convenient manner of administration is generally oral using aconvenient daily dosing regimen which can be adjusted according to theseverity of the disease and the patient's response to the antiviralmedication.

The compounds 4, R_(P)-4, or S_(P)-4 together with one or moreconventional excipients, carriers, or diluents, may be placed into theform of pharmaceutical compositions and unit dosages. The pharmaceuticalcompositions and unit dosage forms may be comprised of conventionalingredients in conventional proportions, with or without additionalactive compounds and the unit dosage forms may contain any suitableeffective amount of the active ingredient commensurate with the intendeddaily dosage range to be employed. The pharmaceutical compositions maybe employed as solids, such as tablets or filled capsules, semisolids,powders, sustained release formulations, or liquids such as suspensions,emulsions, or filled capsules for oral use; or in the form ofsuppositories for rectal or vaginal administration. A typicalpreparation will contain from about 5% to about 95% active compound orcompounds (w/w).

The compounds 4, R_(P)-4, or S_(P)-4 can be administered alone but willgenerally be administered in admixture with one or more suitablepharmaceutical excipients, diluents or carriers selected with regard tothe intended route of administration and standard pharmaceuticalpractice.

Solid form preparations include, for example, powders, tablets, pills,capsules, suppositories, and dispersible granules. A solid carrier maybe one or more substances which may also act as diluents, flavoringagents, solubilizers, lubricants, suspending agents, binders,preservatives, tablet disintegrating agents, or an encapsulatingmaterial. In powders, the carrier generally is a finely divided solidwhich is a mixture with the finely divided active component. In tablets,the active component generally is mixed with the carrier having thenecessary binding capacity in suitable proportions and compacted in theshape and size desired. Suitable carriers include but are not limited tomagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.Solid form preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like. Examples of solid formulations are exemplified in EP 0524579;US 2002/0142050; US 2004/0224917; US 2005/0048116; US 2005/0058710; US2006/0034937; US 2006/0057196; US 2006/0188570; US 2007/0026073; US2007/0059360; US 2007/0077295; US 2007/0099902; US 2008/0014228; U.S.Pat. No. 6,267,985; U.S. Pat. No. 6,294,192; U.S. Pat. No. 6,383,471;U.S. Pat. No. 6,395,300; U.S. Pat. No. 6,569,463; U.S. Pat. No.6,635,278; U.S. Pat. No. 6,645,528; U.S. Pat. No. 6,923,988; U.S. Pat.No. 6,932,983; U.S. Pat. No. 7,060,294; and U.S. Pat. No. 7,462,608,each of which is incorporated by reference.

Liquid formulations also are suitable for oral administration includeliquid formulation including emulsions, syrups, elixirs and aqueoussuspensions. These include solid form preparations which are intended tobe converted to liquid form preparations shortly before use. Examples ofliquid formulation are exemplified in U.S. Pat. Nos. 3,994,974;5,695,784; and 6,977,257. Emulsions may be prepared in solutions, forexample, in aqueous propylene glycol solutions or may containemulsifying agents such as lecithin, sorbitan monooleate, or acacia.Aqueous suspensions can be prepared by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well known suspending agents.

The compounds 4, R_(P)-4, or S_(P)-4 may be independently formulated foradministration as suppositories. A low melting wax, such as a mixture offatty acid glycerides or cocoa butter is first melted and the activecomponent is dispersed homogeneously, for example, by stirring. Themolten homogeneous mixture is then poured into convenient sized molds,allowed to cool, and to solidify.

The compounds 4, R_(P)-4, or S_(P)-4 may be independently formulated forvaginal administration. Pessaries, tampons, creams, gels, pastes, foamsor sprays containing in addition to the active ingredient such carriersas are known in the art to be appropriate. Certain of these formulationsmay also be used in conjunction with a condom with or without aspermicidal agent.

Suitable formulations along with pharmaceutical carriers, diluents andexcipients are described in Remington: The Science and Practice ofPharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19thedition, Easton, Pa., which is hereby incorporated by reference. Askilled formulation scientist may modify the formulations within theteachings of the specification to provide numerous formulations for aparticular route of administration without rendering compositionscontaining the compounds contemplated herein unstable or compromisingtheir therapeutic activity.

Additionally, the purified compounds 4, R_(P)-4, or S_(P)-4 may beindependently formulated in conjunction with liposomes or micelles. Asto liposomes, it is contemplated that the purified compounds can beformulated in a manner as disclosed in U.S. Pat. Nos. 4,797,285;5,013,556; 5,077,056; 5,077,057; 5,154,930; 5,192,549; 5,213,804;5,225,212; 5,277,914; 5,316,771; 5,376,380; 5,549,910; 5,567,434;5,736,155; 5,827,533; 5,882,679; 5,891,468; 6,060,080; 6,132,763;6,143,321; 6,180,134; 6,200,598; 6,214,375; 6,224,903; 6,296,870;6,653,455; 6,680,068; 6,726,925; 7,060,689; and 7,070,801, each of whichis incorporated by reference. As to micelles, it is contemplated thatthe purified compounds can be formulated in a manner as disclosed inU.S. Pat. Nos. 5,145,684 and 5,091,188, both of which are incorporatedby reference.

The fifth embodiment is directed to a use of any of compounds 4,R_(P)-4, or S_(P)-4 in the manufacture of a medicament for the treatmentof any condition the result of an infection by any one of the followingviral agents: hepatitis C virus, West Nile virus, yellow fever virus,degue virus, rhinovirus, polio virus, hepatitis A virus, bovine viraldiarrhea virus and Japanese encephalitis virus.

The term “medicament” means a substance used in a method of treatmentand/or prophylaxis of a subject in need thereof, wherein the substanceincludes, but is not limited to, a composition, a formulation, a dosageform, and the like, comprising any of compounds 4, R_(P)-4, or S_(P)-4.It is contemplated that the use of any of compounds 4, R_(P)-4, orS_(P)-4 in the manufacture of a medicament, for the treatment of any ofthe antiviral conditions disclosed herein, either alone or incombination with another compound disclosed herein. A medicamentincludes, but is not limited to, any one of the compositionscontemplated by the fourth embodiment disclosed herein.

A sixth embodiment is directed to a method of treatment and/orprophylaxis in a subject in need thereof said method comprisesadministering a therapeutically effective amount of any of compounds 4,R_(P)-4, or S_(P)-4 to the subject.

It is intended that a subject in need thereof is one that has anycondition the result of an infection by any of the viral agentsdisclosed herein, which includes, but is not limited to, hepatitis Cvirus, West Nile virus, yellow fever virus, degue virus, rhinovirus,polio virus, hepatitis A virus, bovine viral diarrhea virus or Japaneseencephalitis virus, flaviviridae viruses or pestiviruses orhepaciviruses or a viral agent causing symptoms equivalent or comparableto any of the above-listed viruses.

The term “subject” means a mammal, which includes, but is not limitedto, cattle, pigs, sheep, chicken, turkey, buffalo, llama, ostrich, dogs,cats, and humans, preferably the subject is a human. It is contemplatedthat in the method of treating a subject thereof of the ninth embodimentcan be any of the compounds contemplated herein, either alone or incombination with another compound disclosed herein.

The term “therapeutically effective amount” as used herein means anamount required to reduce symptoms of the disease in an individual. Thedose will be adjusted to the individual requirements in each particularcase. That dosage can vary within wide limits depending upon numerousfactors such as the severity of the disease to be treated, the age andgeneral health condition of the patient, other medicaments with whichthe patient is being treated, the route and form of administration andthe preferences and experience of the medical practitioner involved. Fororal administration, a daily dosage of between about 0.001 and about 10g, including all values in between, such as 0.001, 0.0025, 0.005,0.0075, 0.01, 0.025, 0.050, 0.075, 0.1, 0.125, 0.150, 0.175, 0.2, 0.25,0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, and 9.5, per day should be appropriate in monotherapy and/or incombination therapy. A particular daily dosage is between about 0.01 andabout 1 g per day, including all incremental values of 0.01 g (i.e., 10mg) in between, a preferred daily dosage about 0.01 and about 0.8 g perday, more preferably about 0.01 and about 0.6 g per day, and mostpreferably about 0.01 and about 0.25 g per day, each of which includingall incremental values of 0.01 g in between. Generally, treatment isinitiated with a large initial “loading dose” to rapidly reduce oreliminate the virus following by a decreasing the dose to a levelsufficient to prevent resurgence of the infection. One of ordinary skillin treating diseases described herein will be able, without undueexperimentation and in reliance on knowledge, experience and thedisclosures of this application, to ascertain a therapeuticallyeffective amount of the compound disclosed herein for a given diseaseand patient.

Therapeutic efficacy can be ascertained from tests of liver functionincluding, but not limited to protein levels such as serum proteins(e.g., albumin, clotting factors, alkaline phosphatase,aminotransferases (e.g., alanine transaminase, aspartate transaminase),5′-nucleosidase, γ-glutaminyltranspeptidase, etc.), synthesis ofbilirubin, synthesis of cholesterol, and synthesis of bile acids; aliver metabolic function, including, but not limited to, carbohydratemetabolism, amino acid and ammonia metabolism. Alternatively thetherapeutic effectiveness may be monitored by measuring HCV-RNA. Theresults of these tests will allow the dose to be optimized.

A first aspect of the sixth embodiment is directed to a method oftreatment and/or prophylaxis in a subject in need thereof said methodcomprises administering to the subject a therapeutically effectiveamount of a compound represented by any of compounds 4, R_(P)-4, orS_(P)-4 and a therapeutically effective amount of another antiviralagent; wherein the administration is concurrent or alternative. It isunderstood that the time between alternative administration can rangebetween 1-24 hours, which includes any sub-range in between including,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, and 23 hours.

Examples of “another antiviral agent” include, but are not limited to:HCV NS3 protease inhibitors (see EP 1881001, US 2003187018, US2005267018, WO 2003006490, WO 200364456, WO 2004094452, WO 2005028502,WO 2005037214, WO 2005095403, WO 2007014920, WO 2007014921, WO2007014922, WO 2007014925, WO 2007014926, WO 2007015824, WO 2008010921,and WO 2008010921); HCV NS5B Inhibitors (see US 2004229840, US2005154056, US 2005-98125, US 20060194749, US 20060241064, US20060293306, US 2006040890, US 2006040927, US 2006166964, US 2007275947,U.S. Pat. No. 6,784,166, US20072759300, WO 2002057287, WO 2002057425, WO2003010141, WO 2003037895, WO 2003105770, WO 2004000858, WO 2004002940,WO 2004002944, WO 2004002977, WO 2004003138, WO 2004041201, WO2004065367, WO 2004096210, WO 2005021568, WO 2005103045, WO 2005123087,WO 2006012078, WO 2006020082, WO 2006065335, WO 2006065590, WO2006093801, WO 200702602, WO 2007039142, WO 2007039145, WO 2007076034,WO 2007088148, WO 2007092000, and WO2007095269); HCV NS4 Inhibitors (seeWO 2005067900 and WO 2007070556); HCV NS5a Inhibitors (see US2006276511, WO 2006035061, WO 2006100310, WO 2006120251, and WO2006120252); Toll-like receptor agonists (see WO 2007093901); and otherinhibitors (see WO 2000006529, WO 2003101993, WO 2004009020, WO2004014313, WO 2004014852, and WO 2004035571); and compounds disclosedin U.S. patent application Ser. No. 12/053,015, filed Mar. 21, 2008 (US2010/0016251) (the contents of which are incorporated by reference),interferon-α, interferon-β, pegylated interferon-α, ribavirin,levovirin, viramidine, another nucleoside HCV polymerase inhibitor, aHCV non-nucleoside polymerase inhibitor, a HCV protease inhibitor, a HCVhelicase inhibitor or a HCV fusion inhibitor.

When any of compounds 4, R_(P)-4, or S_(P)-4 are administered incombination with another antiviral agent the activity may be increasedover the parent compound. When the treatment is combination therapy,such administration may be concurrent or sequential with respect to thatof the nucleoside derivatives. “Concurrent administration” as usedherein thus includes administration of the agents at the same time or atdifferent times. Administration of two or more agents at the same timecan be achieved by a single formulation containing two or more activeingredients or by substantially simultaneous administration of two ormore dosage forms with a single active agent.

It will be understood that references herein to treatment extend toprophylaxis as well as to the treatment of existing conditions.Furthermore, the term “treatment” of a HCV infection, as used herein,also includes treatment or prophylaxis of a disease or a conditionassociated with or mediated by HCV infection, or the clinical symptomsthereof.

Preparation

A seventh embodiment is directed to a process for preparing any one ofcompounds 4, R_(P)-4, or S_(P)-4, which comprises: a) reacting anisopropyl-alanate, A, a di-LG-phenylphosphate, B,2′-deoxy-2′-fluoro-2′-C-methyluridine, 3, and a base to obtain a firstmixture comprising at least one of S_(P)-4 and R_(P)-4

wherein X is a conjugate base of an acid, n is 0 or 1, and LG is aleaving group; b) reacting the first mixture with a protecting compoundto obtain a second mixture comprising at least one of protected S_(P)-4and protected R_(P)-4; and c) optionally subjecting the second mixtureto crystallization, chromatography, or extraction in order to obtain 4,S_(P)-4, or R_(P)-4.

In a first aspect of the seventh embodiment, the isopropyl alanate ispresent as its hydrochloric acid salt, which is preferably,substantially anhydrous.

In a second aspect of the seventh embodiment, the base isN-methylimidazole.

In a third aspect of the seventh embodiment, the mole ratio ofA-to-B-to-3 is about 1.6-to-1.3-to-1.

In a fourth aspect of the seventh embodiment, the protecting compound ist-butyl-dimethyl-silyl-chloride.

An eighth embodiment is directed to a process for preparing S_(P)-4 orR_(P)-4, which comprises: a) reacting an isopropyl-alanate, A, adi-LG-phenylphosphate, B, 2′-deoxy-2′-fluoro-2′-C-methyluridine, 3, anda base to obtain a first mixture comprising at least one of S_(P)-4 andR_(P)-4

wherein X is a conjugate base of an acid, n is 0 or 1, and LG is aleaving group; and b) optionally subjecting the second mixture tocrystallization, chromatography, or extraction in order to obtainpurified S_(P)-4 or R_(P)-4.

A first aspect of the eighth embodiment for preparing R_(P)-4additionally includes further purifying the second mixture or thepurified R_(P)-4 by dissolving or suspending the second mixture or thepurified R_(P)-4 mixture in a solvent; optionally followed by seedingwith crystalline R_(P)-4; and adding sufficient anti-solvent to obtaincrystalline R_(P)-4.

A second aspect of the eighth embodiment for preparing S_(P)-4additionally includes further purifying the second mixture or thepurified S_(P)-4 by d) dissolving or suspending the second mixture orthe purified S_(P)-4 in a solvent followed by seeding with crystallineS_(P)-4 at about room temperature; collecting a first solid the majorityof which comprises S_(P)-4; dissolving the first solid in a solvent atits reflux temperature; and cooling or adding an anti-solvent to obtaina second solid.

A third aspect of the eighth embodiment for the preparation of S_(P)-4,additionally includes further purifying S_(P)-4 by d) dissolving orsuspending the second mixture or the purified S_(P)-4 mixture in a firstsolvent followed by adding an anti-solvent so as to obtain a firstcomposition in which the residual solvent/anti-solvent is removed bydecanting to obtain a residue; treating the residue with a solutioncontaining the first solvent and anti-solvent to yield a secondcomposition whereby upon reducing the pressure affords a first solid;dissolving or suspending the first solid using a second solvent so as toobtain a third composition; adding seed crystals of S_(P)-4 to the thirdcomposition; collecting a second solid; dissolving or suspending thesecond solid in a third solvent, optionally heated to the refluxtemperature of the third solvent to obtain a fourth composition, and, ifnecessary, cooling the fourth composition to obtain a third solidcomprising S_(P)-4 which is collected by filtration.

In a fourth aspect of the eighth embodiment for the preparation ofS_(P)-4, S_(P)-4 is further purified by the second mixture or thepurified S_(P)-4 by d) adding silica gel to the second mixture or thepurified S_(P)-4 followed by solvent evaporation to afford a dry slurry;stirring the dry slurry in a first solvent/anti-solvent combination toobtain a first wet slurry; decanting the first solvent/anti-solventcombination from the first wet slurry to obtain a second wet slurry anda first composition; adding to the second wet slurry a secondsolvent/anti-solvent combination followed by stirring; decanting thesecond solvent/anti-solvent combination from the second wet slurry toobtain a third wet slurry and a second composition; optionally repeatingsteps g)-h) on the third wet slurry or additional wet slurries;evaporating the solvent from the second composition, and optionally anyadditional composition obtained from optional step i) to obtain a firstsolid; dissolving or suspending the first solid in a solution containinga third solvent and optionally a fourth solvent to obtain a thirdcomposition; optionally adding seed crystals of S_(P)-4 to the thirdcomposition; obtaining from the third composition a second solidcomprising S_(P)-4; and optionally recrystallizing the second solidusing a third solvent to obtain a third solid comprising S_(P)-4.

One of ordinary skill will appreciate that the compounds can beseparated by traditional extraction, traditional crystallization ortraditional chromatographic techniques. Traditional chromatographictechniques include, but are not limited to, chromatography on silica gel(using, e.g., 3-5% methanol in DCM or 4-6% isopropanol in DCM) toproduce enhanced levels of one isomer (50-100%) and then crystallize it.Alternatively, one could use reversed phase chromatography (using, e.g.,1-30% acetonitrile-aqueous mobile phase). Furthermore the compounds canbe isolated by supercritical fluid chromatography SFC with carbondioxide as the main solvent and alcohols such as methanol as a modifier,preferably using the appropriate chiral media, such as, DaicelChiralpack IA. Alternatively, SMB chromatography may be employed usingthe appropriate chiral media, such as, Daicel ChiralPack IA, using amixture of solvents such as hexanes/isopropanol or single solvents suchas ethyl acetate.

A ninth embodiment is directed to a process for preparing S_(P)-4, whichcomprises: a) reacting an isopropyl-alanyl-phosphoramidate with a3′-O-protected or unprotected 3, and a basic reagent to obtain acomposition comprising protected or unprotected S_(P)-4

wherein the isopropyl-alanyl-phosphoramidate is comprised of a mixtureof diastereomers represented by the following structures:

wherein the ratio of C:C′ is about 1:1.

In a first aspect, the basic reagent is t-butylmagnesium chloride andthe ratio of C:C′ is greater than or equal to about 1:1.

In a second aspect, the basic reagent is t-butylmagnesium chloride andthe ratio of C:C′; is greater than about 1:1.

In a third aspect, the basic reagent is t-butylmagnesium chloride andthe ratio of C:C′ is at least about 1.5:1, about 2.3:1, about 4:1, about5.7:1, about 9:1, about 19:1, about 32.3:1, about 49:1, or about 99:1.

A fourth aspect the LG′ is p-nitrophenoxide, the basic reagent ist-butylmagnesium chloride, and the ratio of C:C′ is at least about1.5:1, about 2.3:1, about 4:1, about 5.7:1, about 9:1, about 19:1, about32.3:1, about 49:1, or about 99:1.

A fifth aspect for preparing S_(P)-4, comprises: a) reacting anisopropyl-alanyl-phosphoramidate (C) with a 3′-O-protected orunprotected 3, and a basic reagent to obtain a composition comprisingprotected or unprotected S_(P)-4

wherein Z is a protecting group or hydrogen; LG′ is a leaving group; andb) optionally subjecting the obtained protected or unprotected S_(P)-4to chromatography, extraction, or crystallization in order to obtainpurified protected or unprotected S_(P)-4. In a sub-embodiment, LG′ istosylate, camphorsulfonate, or an aryloxide substituted with at leastone electron withdrawing group; more preferably, LG′ is selected fromamong p-nitrophenoxide, 2,4-dinitrophenoxide, and pentafluorophenoxide.In a further sub-embodiment, when S_(P)-4 is protected, i.e., Z is nothydrogen, the process of the ninth embodiment is further directed todeprotecting protected S_(P)-4. In a further sub-embodiment, thereaction is conducted in a polar aprotic solvent, such as,tetrahydrofuran or another etheral solvent either one being alone or incombination with each other or with a C₂ to C₇ nitrile, such asacetonitrile.

The process of the ninth embodiment further comprises 1) reacting(LG′)P(O)(LG)₂, wherein LG, independent of LG′, is a leaving group, with(i) isopropyl-alanate and a first base to obtain(LG′)P(O)(LG)(NHAla-^(i)Pr) followed by reacting(LG′)P(O)(LG)(NHAla-^(i)Pr) with phenol and a second base to obtain amixture comprising C and C′, (ii) phenol and a first base to obtain(LG′)P(O)(LG)(OPh) followed by reacting (LG′)P(O)(LG)(OPh) withisopropyl-alanate and a second base to obtain a mixture comprising C andC′, or (iii) combining isopropyl-alanate, phenol, and at least one baseto obtain a mixture comprising C and C′; or 2) reacting (PhO)P(O)(LG)₂,wherein LG′, independent of LG, is a leaving group, with (i)isopropyl-alanate and a first base to obtain (PhO)P(O)(LG)(NHAla-^(i)Pr)followed by reacting (PhO)P(O)(LG)(NHAla-^(i)Pr) with a leaving groupprecursor and a second base to obtain a mixture comprising C and C′,

and subjecting the mixture to chromatography or crystallizing themixture to obtain C. In an aspect of the ninth embodiment, the isopropylalanate is present as its hydrochloric acid salt, which is preferably,substantially anhydrous.

A tenth embodiment is directed to a process for preparing R_(P)-4, whichcomprises: a) reacting an isopropyl-alanyl-phosphoramidate with a3′-O-protected or unprotected 3, and a basic reagent to obtain acomposition comprising protected or unprotected R_(P)-4

wherein the isopropyl-alanyl-phosphoramidate is comprised of a mixtureof diastereomers represented by the following structures:

wherein the ratio of C′:C is about 1:1.

In a first aspect, the basic reagent is t-butylmagnesium chloride andthe ratio of C′:C is greater than or equal to about 1:1.

In a second aspect, the basic reagent is t-butylmagnesium chloride andthe ratio of C′:C; is greater than about 1:1.

In a third aspect, the basic reagent is t-butylmagnesium chloride andthe ratio of C′:C is at least about 1.5:1, about 2.3:1, about 4:1, about5.7:1, about 9:1, about 19:1, about 32.3:1, about 49:1, or about 99:1.

A fourth aspect the LG′ is p-nitrophenoxide, the basic reagent ist-butylmagnesium chloride, and the ratio of C′:C is at least about1.5:1, about 2.3:1, about 4:1, about 5.7:1, about 9:1, about 19:1, about32.3:1, about 49:1, or about 99:1.

A fifth aspect for preparing R_(P)-4, comprises: a) reacting anisopropyl-alanyl-phosphoramidate (C′) with a 3′-O-protected orunprotected 3, and a basic reagent to obtain a composition comprisingprotected or unprotected R_(P)-4

wherein Z is a protecting group or hydrogen; LG′ is a leaving group; andb) optionally subjecting the obtained protected or unprotected R_(P)-4to chromatography, extraction, or crystallization in order to obtainpurified protected or unprotected R_(P)-4. In a sub-embodiment, LG′ istosylate, camphorsulfonate, or an aryloxide substituted with at leastone electron withdrawing group; more preferably, LG′ is selected fromamong p-nitrophenoxide, 2,4-dinitrophenoxide, and pentafluorophenoxide.In a further sub-embodiment, when R_(P)-4 is protected, i.e., Z is nothydrogen, the process of the ninth embodiment is further directed todeprotecting protected R_(P)-4. In a further sub-embodiment, thereaction is conducted in a polar aprotic solvent, such as,tetrahydrofuran or another etheral solvent either one being alone or incombination with each other or with a C₂ to C₇ nitrile, such asacetonitrile.

The process of the tenth embodiment further comprises 1) reacting(LG′)P(O)(LG)₂, wherein LG, independent of LG′, is a leaving group, with(i) isopropyl-alanate and a first base to obtain(LG′)P(O)(LG)(NHAla-^(i)Pr) followed by reacting(LG′)P(O)(LG)(NHAla-^(i)Pr) with phenol and a second base to obtain amixture comprising C and C′, (ii) phenol and a first base to obtain(LG′)P(O)(LG)(OPh) followed by reacting (LG′)P(O)(LG)(OPh) withisopropyl-alanate and a second base to obtain a mixture comprising C andC′, or (iii) combining isopropyl-alanate, phenol, and at least one baseto obtain a mixture comprising C and C′; or 2) reacting (PhO)P(O)(LG)₂,wherein LG′, independent of LG, is a leaving group, with (i)isopropyl-alanate and a first base to obtain (PhO)P(O)(LG)(NHAla-^(i)Pr)followed by reacting (PhO)P(O)(LG)(NHAla-^(i)Pr) with a leaving groupprecursor and a second base to obtain a mixture comprising C and C′,

and subjecting the mixture to chromatography or crystallizing themixture to obtain C′. In an aspect of the ninth embodiment, theisopropyl alanate is present as its hydrochloric acid salt, which ispreferably, substantially anhydrous.

An eleventh embodiment is directed to a composition obtained by theprocesses recited in the seventh embodiment, the eighth embodiment, theninth embodiment or the tenth embodiment as well as their respectiveaspects. An aspect of the eleventh embodiment is directed to acomposition obtained by any one of the exemplified embodiments disclosedbelow. The so obtained composition can be crystalline, crystal-like,amorphous, or a combination thereof.

A twelfth embodiment is directed to a compound 3

wherein Z is a protecting group or hydrogen; which is useful for thepreparation of R_(P)-4 or S_(P)-4.

A first aspect of the twelfth embodiment is selected from among acompound having the following structure

A thirteenth embodiment is directed to a compound, its salt, hydrate,solvate, or combination thereof, represented by the following structures

where LG′ is a leaving group, which is useful for the preparation ofR_(P)-4 or S_(P)-4.

In a first aspect of the thirteenth embodiment, LG′ is tosylate,camphorsulfonate, an aryloxide, or an aryloxide substituted with atleast one electron withdrawing group.

In a second aspect of the thirteenth embodiment, LG′ is selected fromamong p-nitrophenoxide, 2,4-dinitrophenoxide, and pentafluorophenoxide.

A fourteenth embodiment is directed to an isotopically-labeled analog ofR_(P)-4 or S_(P)-4. The term “isotopically-labled” analog refers to ananalog of R_(P)-4 or S_(P)-4 that is a “deuterated analog”, a“¹³C-labeled analog,” or a “deuterated/¹³-C-labeled analog.” The term“deuterated analog” means a compound described herein, whereby a¹H-isotope, i.e., hydrogen (H), is substituted by a ²H-isotope, i.e.,deuterium (D). Deuterium substitution can be partial or complete.Partial deuterium substitution means that at least one hydrogen issubstituted by at least one deuterium. For instance, for R_(P)-4 orS_(P)-4, one of ordinary skill can contemplate at least the followingpartial deuterated analogs (where “d_(n)” represents n-number ofdeuterium atoms, such as, for an isopropyl group n=1-7, while for aphenyl group, n=1-5), as well as those depicted below.

Although the methyl groups depicted above are shown as being completelydeuterated, one will recognize that partial-deuterated variations arealso possible, such as, —CDH₂ and —CD₂H. Isotopic labels on the furanoseand base are also contemplated. Likewise, the terms “¹³C-labeled analog”and “deuterated/¹³-C-labeled analog” refers to a compound describedherein, whereby carbon atom is enriched with a ¹³C-isotope meaning thatthe extent of enrichment exceeds the usual natural abundance of about1.1%.

EXAMPLES

Not to be limited by way of example, the following examples serve tofacilitate a better understanding of the disclosure.

Synthetic Aspects

In order to prepare the uridine nucleoside, one could take advantage ofan advanced tribenzoylated cytidine intermediate in the synthesis ofcertain 3′,5′-diacylated analogs of 3 (see below) already producedefficiently on a pilot plant scale (see WO 2006/031725 or US2006/0122146, both of which are incorporated by reference in theirentirety). The following method was found to be scalable andcost-efficient.

3′,5′-O-dibenozyl-2′-deoxy-2′-fluoro-2′-C-methyl-N⁴-benzoylcytidine (1)is obtained by a method disclosed in WO 2006/031725 and WO 2008/045419both of which are hereby incorporated by reference in its entirety. 1 istreated with 70% aqueous acetic acid to form3′,5′-O-dibenozyl-2′-deoxy-2′-fluoro-2′-C-methyl-uridine (2). Thebenzoyl esters can be hydrolyzed by a number of methods as well, e.g.,alkoxides in alcoholic solvent, such as sodium methoxide in methanol,potassium carbonate in methanol, or ethanol analogs, alkylamines such asmethylamine in methanol, butylamine etc. Methanolic ammonia was chosenfor the larger scale work. The uridine product (3) can be purified bycrystallization to afford a 70% yield from the tribenzoylated cytidine(1).

Numerous literature procedures detail different routes and conditions tomake phosphoramidates using several fold equivalents of reagents. See,for example, McGuigan et al. J. Med. Chem. 2005, 48, 3504-3515 andMcGuigan et al. J. Med. Chem. 2006, 49, 7215. For process scale work,there is only one presently known example, which is disclosed in Lehstenet al., Org. Process Res. Dev. 2002, 6, 819-822 (“Lehsten”). In thisreference, the authors introduce the concept of a “one-pot procedure” inwhich an amino acid hydrochloride salt and phenyl dichlorophosphate arereacted together with N-methylimidazole in dichloromethane. Later thenucleoside is added to form the desired 5′-O-phosphoramidate product,which in the present case would yield a compound represented by formula4. Unfortunately, the Lehsten procedure suffered from drawbacks. Forexample, the Lehsten procedure utilized a far larger excess of reagentsthan was necessary which added to the cost and difficulty ofchromatographic purification. Furthermore, Lehsten suggested that onecould control the reaction selectivity on the 5′-hydroxyl over the3′-hydroxyl compared to a literature reference through using lowertemperatures and slow addition of the nucleoside.

Using the Lehsten procedure for the compounds disclosed herein providedfor about 1-5% of mono-substituted 3′-O-phosphoramidate diastereomers(5) and about 10-30% of the bis-substituted product (6). As the polarityof the 3′-diastereomers was very similar to the desired 5′-diastereomers(4), chromatographic separation was very challenging. Scaling up theprocess was nearly impossible without discarding a substantial portionof the less polar 5′-diastereomers (4) or accepting a higher level ofcontamination of the 3′-diastereomers (5). In an initial 50 g scale-up,the resultant product contained a 3′-diastereomer (5) contamination ofabout 3%, which co-eluted with the less polar of the 5′-diastereromer(4).

Disclosed herein are reaction conditions which use lesser amounts ofreagents and a method to selectively remove the impurity3′-O-phosphoramidate diastereomers (5) with an easier chromatographicseparation thereby affording the desired 5′-β-phosphoramidatediastereomers in much higher purity (4).

For the reagent stoichiometry, a study was made in which thestoichiometry of the reagents was systematically changed and the resultswere monitored by phosphorus NMR of the crude reaction as Lehsten hadreported. In the more successful runs, the isolated yield and purity ofthe desired product were compared. It was observed that the primary5′-hydroxyl reacts at a faster rate than the secondary 3′-hydroxyl. Thiscreates a competing situation between the reaction progress of consumingall the starting nucleoside and converting 5′- and 3′-monosubstitutedproducts (4 and 5) to the 5′,3′-bis substituted products (6). The3′-monosubstituted product converts to the bis product at a faster ratethan the 5′-monosubstituted product, so it is possible to reduce the3′-diastereomer contamination level by pushing the reaction more to thebis-substituted products. However, with an effective way to remove the3′-diastereomers, the reaction can be optimized to produce more of thedesired 5′-diastereomer without having to sacrifice as much of the5′-diastereomer being converted to the bis-substituted (6). It was alsoobserved that the amino acid hydrochloride is very hygroscopic. As anywater present would consume an equivalent amount of the phenyldichlorophosphate reagent, care must be taken to keep the amino acidsubstantially anhydrous or it should be made substantially anhydrousprior to use. In short, Lehsten had reported that the optimum ratio ofamino acid to phenyl dichlorophosphate to nucleoside was 3.5:2.5:1respectively. It was found that the optimum ratio of amino acid tophenyl dichlorophosphate to nucleoside of about 1.6 to about 1.3 toabout 1 is optimal under conditions in which the 3′-diastereomer can beefficiently removed and when the amino acid hydrochloride issubstantially anhydrous. By using a smaller amount of the reagents, acost savings is realized coupled with a simplification of thechromatographic separation of the desired product from reagentby-products and from the reduced level of bis diastereomers. In onealternative procedure, a 3′-hydroxy-blocked derivative of 3 was preparedusing a t-butyldimethylsilyl blocking group in two steps. This was thenconverted to its 5′-phosphoramidate derivative. The desire being thatthe silyl group could then be removed and there would be no 3′ isomers(5) or 3′,5′-bis phosphoramidates (6). A similar approach wasdemonstrated by Borch and Fries (U.S. Pat. No. 5,233,031) in a lowoverall yield on an alkyl phosphoramidate.

Another alternative approach was to use the direct synthesis and thenuse chemistry to help differentiate the 3′-diastereomer impurities 5from the desired 5′-diastereomers 4 to help the separation. A group wasdesired that would selectively react with the free primary hydroxyl ofthe 3′-O-phosphoramidate impurity 5 over the free secondary hydroxyl ofthe desired 5′-O-phosphoramidate 4. It was also desired that theblocking group significantly change the polarity of the resulting5′-O-blocked 3′-O-phoshoramidate product from the desired5′-O-phosphoramidate 4. There would be no extra step needed to removethe blocking group as the desired 5′-diastereomers 4 would not bechanged. The chemically altered 3′-diastereomers would then allow easierchromatographic separation or separation by special scavenging supportsor by extractions.

Specifically, the blocking group tert-butyldimethylsilyl (tBDMS) metthese criteria and was the first one to be demonstrated and subsequentlyused on a multi-kilogram scale. Under certain conditions such as inpyridine as solvent and base, the tBDMS group reacts with highselectively at the primary hydroxyl position over the 3′ secondaryhydroxyl position. The phosphoramidate reaction uses N-methylimidazole(NMI) as a base. In the presence of NMI, the silylation is lessselective. Preferably, the amount of NMI should be reduced. This can beaccomplished easily after the phosphoramidate reaction by washing thereaction solution with 1 N hydrochloric acid. The NMI and the remainingstarting nucleoside are removed, leaving a crude mixture of mono and bissubstituted products and reagent by-products. This is then dissolved inpyridine and treated with tert-butyldimethylsilyl chloride. The3′-monosubstituted product 5 is converted in a few hours or less to the5′-O-tBDMS-3′-O-phosphoramidate 7. The reaction progress can bemonitored by HPLC. The polarity of this silylated product 7 is less thanthe bis-phosphoramidate 6 and is readily removed by chromatography.Using this method, it was possible to reduce the level of3′-monophosphoramidate 5 to less than 0.1% of the 5′-product 4 comparedto 1-3% without the silyl treatment. Similarly, treatment withdimethoxytriphenylmethyl chloride (DMT-Cl) under the same conditionsworked just as well. It was also easier to identify the DMT reactionproduct by TLC as DMT containing molecules stain bright orange onheating or exposure to acid. One can also envision many other blockinggroups, as noted above.

Both the reaction conditions and the scavenging of the 3′-impurity aregeneral methods and could be applied to most nucleoside phosphoramidateswith a free 3′ hydroxyl. The phosphoramidate moiety could be anycombination of amino acid ester and aromatic alcohol. The nucleosidemoiety could be any nucleoside in which a 5′ phosphoramidate would leadto a 5′-monophosphate and could be further metabolized to the5′-triphosphate form.

The following scheme is the main reaction scheme illustrated for makingisopropyl L-alanate phenyl phosphoramidate of2′-deoxy-2′-fluoro-2′-C-methyluridine with the major product as thedesired 5′-O-phosphoramidate (4, two diastereomers) and the minorproduct as the 3′-O-phosphoramidate (5, two diastereomers) and the3′,5′-bis-O-phosphoramidate (6, four diastereomers). The reagents areadded in the stoichiometric ratios as described in the method ofpreparation section. The reaction is allowed to proceed until about 5%of the starting material remains as judged by UV visualization on thinlayer chromatography (TLC). Also UPLC/MS showed approximately 10% of the3′,5′ bis-phosphoramidate 6 had formed compared to the desired5′-product. After quenching and an acidic aqueous workup, the cruderesidue from the organic layer was prepared for the silylation. Underthe described reaction conditions, the silyl group preferentiallyreacted with the free 5′-hydroxyl of the 3′-O-phosphoramidate to form 7.The reaction was continued until the 3′-O-phosphoramidate was no longerdetectable by UPLC/MS.

After working up the silylation reaction, the desired product issubjected to chromatography on silica gel and is eluted with a gradientof methanol in dichloromethane (1-4%). The desired5′-monophosphoramidate 4 elutes last.

Method of Preparation Example 1 Preparation of2′-deoxy-2′-fluoro-2′-C-methyluridine (3)

In a 10 L flask, was added3′,5′-O-dibenozyl-2′-deoxy-2′-fluoro-2′-C-methyl-N4-benzoylcytidine (500g, 0.874 mol) and 70% aqueous acetic acid (7.5 L). The solution washeated to reflux (110° C.) for 20 h. TLC indicated a complete reaction(Rf 0.6 in 5% methanol in dichloromethane (DCM)). The mixture was cooledto ambient temperature and diluted with water (2 L). After stirring for2 h, the resulting precipitate was collected by filtration and the solidwas rinsed with water (5 L) and dried in the atmosphere at ambienttemperature for 12 h to afford 360 g (88%). This dibenzoyluridineintermediate was used directly in the next step by adding it all tofreshly prepared methanolic ammonia (5.4 L, ca 25%) at 0° C. Thistemperature was maintained for 3 h and then allowed to warm to 15° C.for 24 h. TLC indicated a complete reaction (R_(f) 0.4 in 10% methanolin DCM). The reaction mixture was filtered through a Celite bed andconcentrated under reduced pressure to give the crude product (216 g).The crude product was stirred with ethyl acetate (325 mL) for 3 h atambient temperature. The resulting solid was collected by filtration andwashed with ethyl acetate (216 mL). The solid was dried under vacuum atambient temperature for 4 h to afford 160 g (78%) of the desired productin 98.7% HPLC purity. ¹H-NMR (DMSO-d₆) δ 11.44 (br s, 1H, NH), 7.95 (d,1H, C-6H), 5.97 (d, 1H, C-1′H), 5.64 (d, 1H, C-5H), 3.84-3.77 (m, 3H,C-5′-Ha, C-3′H. C-4′H), 3.63-3.60 (m, 1H, C5′-Hb), 1.23 (d, 3H,C-2′-CH₃). ES-MS M-1 259.

Example 2 Preparation of(S)-2-{[(1R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-4-(R)-fluoro-3-hydroxy-4-methyl-tetrahydro-furan-2-ylmethoxy]-phenoxy-phosphorylamino}-propionicacid isopropyl ester (4)

Synonym: 5′-O-(Isopropyl-L-alanate, phenylphosphoramidyl)-2′-deoxy-2′-fluoro-2′-C-methyl-uridine diastereomericmixture.

A 5 L 3-necked flask was fitted with a mechanical stirrer, brine icebath, internal thermometer, and a nitrogen atmosphere. The flask wascharged with L-alanine isopropyl ester hydrochloride (82.0 g, 0.490moles) and anhydrous dichloromethane (0.80 L). While this was stirring,phenyl dichlorophosphate (85.0 g, 0.40 moles) was added in one lot andstirred. While maintaining the internal temperature between −5 to 5° C.,a solution of N-methylimidazole (NMI, 250 g, 3.07 moles) indichloromethane (250 mL) was added over a period of a half hour. Thesolution was allowed to stir for 1 h in this temperature range.2′-Deoxy-2′-fluoro-2′-C-methyl-uridine (3, 80.0 g, 0.307 moles) wasadded at 0° C. in one portion and then the reaction flask was allowed towarm up slowly in the brine bath. At 1 h, the internal temperature wasup to −2° C. TLC (5% Methanol in DCM) at 1 h showed that more than 50%of nucleoside was consumed. The bath was removed and the reaction flaskreached ambient temperature over 1 h more. TLC after 3 h and at 5 htotal showed 95% of the starting nucleoside was consumed. The reactionmixture was quenched by adding methanol (100 mL) and stirring thereaction for 5 minutes.

The reaction mixture was washed with 1N HCl (2×500 mL) followed bysaturated sodium bicarbonate solution (2×500 mL). The separated organiclayer was dried over anhydrous sodium sulfate (50 g) and filtered. Thesolution was evaporated under reduced pressure and then under highvacuum to dryness to give the crude product as a viscous oil (170 g).NMRs of the crude product (³¹P and ¹H) were taken. The ³¹P-NMR indicatedabout 1% of the total phosphorus integration was due to the presence ofthe 3′ isomer 5.

To the crude product was added anhydrous pyridine (1700 mL). The solventwas evaporated under reduced pressure and then under high vacuum inorder to reduce the water content of the crude mixture throughco-evaporation. The resulting oil was redissolved in anhydrous pyridine(500 ml) and then was added excess t-butyldimethylsilyl chloride (9.0 g,60 mM). The reaction was stirred at ambient temperature. Reactionprogress was monitored by UPLC/MS. After 3 hours, the 3′ impurity 5could no longer be detected and the reaction was quenched by theaddition of methanol (50 mL).

The reaction was evaporated under reduced pressure to an oil. Theresidue was dissolved in ethyl acetate (1.5 L) and washed with 1N HCl(2×500 mL), followed by saturated sodium bicarbonate solution (2×500mL). The organic layer was dried over anhydrous sodium sulfate (50 g),filtered and evaporated under reduced pressure to give the crude productas a pale yellow oil.

The crude oil was diluted with the same volume of dichloromethane andloaded onto a 2.5 Kg silica gel cartridge in a radial compression moduleat 100 psi of air pressure. Using a gradient pump at 60 psi and a flowrate of 400 mL/min, the cartridge was washed with methylene chloride (4L) followed by a gradient 1-4% methanol in methylene chloride (48 L).Most of the major impurities (di-(isopropylalanyl)phenyl phosphate,3′,5′-bis phosphoramidate (6), 3′-phosphoramidate-5′-TBDMS adduct (7))eluted with ˜3% gradient. The desired product eluted between 3 and 4%methanol. The product containing fractions were sorted into two lots.The first contained small amounts of upper impurities and the latter waspure product. The first set of fractions contained small amounts of lesspolar impurities (upper impurities) such as the 3′,5′-bisphosphoramidate and the di-alanylphenyl phosphate and a mostly the Rpdiastereomer and required a second column purification. (The relativeterminology, upper vs. lower refers to the elution on normal-phasesilica-gel chromatography, where the “upper isomer” means the firsteluting isomer.) The second set of fractions did not have a significantamount of impurities—just the remaining Rp and mostly the Spdiasterereomers. It was later recombined with the twice-columnedfractions. The solvent was evaporated under reduced pressure and theresulting white foam was further dried (0.20 mmHg) for 1 h to give 42 gof the impure lot (4:1 upper vs lower isomer based of ³¹P-NMR) and 38 gof the pure lot (1:3 upper vs lower isomer). The impure lot wasrecolumned in a similar manner to give 3.8 g of 97% pure upper isomer(fraction set aside) and 36 g of pure product in a 4:1 ratio. The twomain lots were dissolved in DCM, combined, evaporated under reducedpressure and dried (50° C., 0.2 mmHg, 24 h) to get 74 g (45.7%) of pureproduct 4 with a diastereomeric ratio of 48:51, as a white foam, mpabout 75-85° C.

In order to produce an amorphous solid of the diastereomeric mixture, 74g of the white foam was stirred in with t-butyl methyl ether (750 mL)resulting in a partial solution and a gummy solid residue. Whilestirring, heptanes (750 mL) was added slowly and the suspension wasmechanically stirred for 1 hour until most of the gum was converted to awhite solid. The solid was scraped up with a spatula and the resultingslurry was filtered. The solid was washed with heptanes (4×50 mL) anddried under vacuum (50° C., 0.2 mmHg, 24 h) to give a white, amorphouspowder (64 g) with a broad melting range of ca 70-80° C. ¹H and ³¹P NMRconformed to structure and HPLC showed a purity of 99.8% with adiastereomeric ratio of 46:54 (also confirmed by ³¹P NMR).

Alternative method to make solid mixture of 4. After chromatography, theresidue was co-evaporated with dichloromethane twice (5 mL/g) and driedfor 24 h at 35-40° C. at 35-45 mTorr. The foam residue was sievedthrough a 250 micron screen and further dried under the same conditionsuntil the residual dichloromethane fell below 400 ppm as measured byheadspace GC. The resulting fine off-white to white amorphous powder hasa glass transition temperature range of 53.7 to 63.5° C.

Characterization of the mixture of isomers (4): ¹H-NMR (CDCl₃) δ 10.05(br s, 1H, NH, S_(P)), 10.00 (br s, 1H, NH, R_(P)), 7.49 (d, 1H, C6-H,S_(P)), 7.36 (m, 5H, C6-H, R_(P), aromatic), 7.23-7.14 (m, 6H,R_(P)/S_(P), aromatic), 6.18 (br d, 2H, C1′-H, R_(P)/S_(P)), 5.63 (d,1H, C5-H, S_(P)), 5.58 (d, 1H, C5-H, R_(P)), 5.01 (m, 2H, CH—(CH₃)₂,R_(P)/S_(P)), 4.46-4.33 (m, 8H, C-5′-H₂, ala-NH, C3′-OH, Rp/S_(P)), 4.12(m, 2H, ala-CH—CH₃, R_(P)/S_(P)), 4.01-3.85 (m, 4H, C3′-H, C4′-H,Rp/S_(P)), 1.39-1.22 (m, 12H, all CH₃, Rp/S_(P)).

³¹P-NMR (CDCl₃) δ 3.60 (R_(P)), 3.20 Sp relative to triphenylphosphateat −17.80 ppm. ES-MS M+1 530.2. Elemental Analysis: Calculated %(including 0.29% water as found by Karl Fisher analysis) C, 49.75; H,5.54; N, 7.90, F, 3.58, P, 5.84. Found %: C, 49.50; H, 5.44; N, 7.85; F,3.62; P, 6.05.

Discussion on Separation of Isomers

Compound 4 due to the chirality at phosphorus is comprised of twodiastereomers, which are designated as S_(P)-4 and R_(P)-4. Thestereochemical assignment was made based on single crystal X-rayanalysis of S_(P)-4. Both R_(P)-4 and S_(P)-4 gave crystalline product.

The procedures for crystallization are outlined below.

Example 3

Crystallization of the R_(P)-4 isomer. The chromatographed fraction ofcontaining the first eluting, less polar R_(P)-4 isomer (3.8 g, 97%pure) was dissolved in isopropanol (36 g) and diluted with heptanesuntil cloudy (72 g). The solution was seeded and stirred at ambienttemperature for 5 h. The resulting solid was collected by vacuumfiltration, washed with heptanes (2×20 mL) and dried (50° C., 0.2 mm, 24h) to 2.3 g of very small white needles mp 136.2-137.8° C. HPLC purityof the resultant material was found to be 99.02%.

R_(P)-4: ¹H-NMR (CDCl₃) δ 9.10 (br s, 1H, NH), 7.36 (m, 2H, o-aromatic),7.26-7.16 (m, 4H, C6-H, m,p-aromatic), 6.16 (br d, 1H, C1′-H), 5.58 (d,1H, C5-H), 5.01 (sept, 1H, CH—(CH₃)₂), 4.52-4.47 (m, 2H, C-5′-H₂), 4.10(d, 1H, C3′-H), 4.02-3.76 (m, 4H, ala-NH, C3′-OH, C4′-H, ala-CH—CH₃),1.37-1.20 (m, 12H, all CH₃).

Example 4 Preparation and Crystallization of S_(P)-4

Method 1: Direct precipitation from crude 4: To a stirred solution ofL-alanine isopropyl ester hydrochloride (10.5 g, 61.5 mmol,azeotropically dried, two times, with 50 mL of toluene each time) indichloromethane (100 mL) was added phenydichlorophosphate (7.5 mL, 50mmol) at room temperature. The mixture was cooled to −10° C. and thenwas added a solution of NMI (30.5 mL, 384.3 mmol) in 30 mL ofdichloromethane over a period of 30 min. After completion of theaddition, the mixture was stirred between −10 and −15° C. for 1 h. Tothe above mixture was added 2′-deoxy-2′-fluoro-2′-C-methyluridine (3)(10 g, 38.4 mmol) in one lot and the mixture was stirred below −10° C.for 3 h and then slowly allowed to warm to 20° C. (6 h). The mixture wasstirred at this temperature over night (15 h) and then quenched with 10mL of methanol. The solvent was evaporated and the residue wasre-dissolved in EtOAc (200 mL). The EtOAc layer was washed with water(100 mL), 1N HCl (3×75 mL), 2% aqueous NaHCO₃ solution (50 mL) and brine(50 mL). The organic layer was dried over Na₂SO₄, filtered andconcentrated. The residue was dried under high vacuum for 2 h to givewhite foam (22 g).

The above foam was dissolved in 33 mL of DCM and then was added 65 mL ofIPE (isopropyl ether) to give a saturated solution. The solution wasfiltered though a small pad of Celite and the filtrate was stirred withS_(P)-4 seeds for 72 h at ambient temperature (about 22° C.—note thatcooling the suspension to 0° C. led to oiling out the crude product).The white solid was filtered, washed with IPE (20 mL) and dried to give4.58 g (˜85:15 mixture of S_(P)-4:R_(P)-4 respectively as determined by³¹P NMR) of a white powder. The above solid was suspended in 23 mL ofDCM and then refluxed for 3 h. The mixture was cooled to roomtemperature and stirred for 15 h. The white solid was filtered, washedwith 4.5 mL of cold DCM and dried under high vacuum at 45° C. to givepure S_(P)-4, mp 93.9-104.7° C., HPLC purity 99.74% (3.11 g, 15.2% fromthe uridine nucleoside).

S_(P)-4 ¹H-NMR (CDCl₃) δ 8.63 (br s, 1H, NH), 7.47 (d, 1H, C6-H), 7.30(m, 2H, o-aromatic), 7.26-7.18 (m, 3H, m,p-aromatic), 6.18 (br d, 1H,C1′-H), 5.70 (d, 1H, C5-H), 5.02 (sept, CH—(CH₃)₂), 4.53 (m, 2H,C-5′-H₂), 4.11 (d, 1H, C3′-H), 3.97 (m, 3H, C3′-OH, C4′-H, ala-CH—CH₃),3.77 (br s, 1H, ala-NH), 1.39 (d, 3H, C2′-CH₃), 1.37 (d, 3H, ala-CH₃),1.24 (d, 6H, CH—(CH₃)₂).

Method 2: Oiling out from crude 4: To a stirred solution of L-alanineisopropyl ester hydrochloride (20.6 g, 123 mmol, azeotropically dried,two times, with 75 mL of toluene each time) in dichloromethane (200 mL)was added phenydichlorophosphate (14.9 mL, 100 mmol) at roomtemperature. The mixture was cooled to −10° C. and then was added asolution of NMI (61.3 mL, 769 mmol) in 60 mL of dichloromethane over aperiod of 30 min. After completion of the addition, the mixture wasstirred between −10° C. and −15° C. for 1 h. To the above mixture wasadded 2′-deoxy-2′-fluoro-2′-C-methyluridine (3) (20 g, 76.9 mmol) in onelot and the mixture was stirred below −10° C. for 3 h and then slowlyallowed to warm to 20° C. (6 h). The mixture was stirred at thistemperature over night (15 h) and then quenched with 10 mL of methanol.The solvent was evaporated and the residue was re-dissolved in EtOAc(400 mL). The EtOAc layer was washed with water (200 mL), 1N HCl (3×100mL), 2% aqueous NaHCO₃ solution (100 mL) and brine (50 mL). The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue wasdried under high vacuum for 2 h to give white foam (43 g). The abovefoam was dissolved in 86 mL of EtOAc in a two neck round bottom flaskequipped with a mechanical stirrer. While stirring, 100 mL of heptanewas added slowly and the suspension was stirred for 1 h. The top layerwas decanted and the residue was again stirred with 50 mL of 2:3EtOAc/heptane solutions for 10 min and then decanted. The residue wasdried under high vacuum to give white foam (31 g).

The above foam was dissolved in 46 mL of DCM and then was added 95 mL ofIPE to give a saturated solution. The solution was filtered though asmall pad of Celite and the filtrate was stirred with S_(P)-4 seeds for72 h at ambient temperature. The white solid was filtered, washed withIPE (30 mL) and dried to give 7.33 g (˜85:15 mixture of S_(P)-4: R_(P)-4respectively as determined by ³¹P NMR) of white powder. The above solidwas suspended in 36 mL of DCM and then refluxed for 3 h. The mixture wascooled to room temperature and stirred for 15 h. The white solid wasfiltered, washed with 7.5 mL of cold DCM and dried under high vacuum at45° C. to give >99% pure S_(P)-4, (4.78 g, 11.6% from the uridinenucleoside).

Method 3: Silica gel loading of crude 4: 5.0 g of crude 4 was producedas in the same manner as the mixture of diastereomers just before thecolumn chromatography step starting with approximately 2.5 g of2′-deoxy-2′-fluoro-2′-C-methyluridine (3). The crude was dissolved in 10mL of DCM and 10 g of silica gel was added to the solution. The solventwas evaporated to give dry slurry. The slurry was stirred with 40 mL of50% EtOAc/hexanes for 15 min and then filtered. The silica gel waswashed with additional 10 mL of 50% EtOAc/hexanes. The silica gel wasthen washed with 15% MeOH/DCM (100 mL) and collected separately. Thesolvent was evaporated and dried under high vacuum to give 4.0 g ofresidue (foam). The residue was dissolved in DCM (6 mL) and then wasadded ˜9 mL of IPE to make a saturated solution. The mixture was thengently stirred overnight with S_(P)-4 seeds at ambient temperature. Thewhite solid was filtered and washed with IPE (5 mL) to give 1.28 g ofproduct. ³¹P NMR revealed that the above product contains 77:23 mixtureof S_(P)-4: R_(P)-4 respectively. This was recrystallized from 20 mL ofDCM to obtain 0.75 g of >99% pure S_(P)-4 (about 12% from the uridinenucleoside). This preparation of S_(P)-4 does not require the silylationstep as done for the mixture, so the entire reaction procedure is shownabove. Aspects of single crystalline and polymorphic forms of S_(P)-4are presented below.

Method 4: 40.0 g of 1:1 mixture of 4 was dissolved in 90 mL ofdichloromethane. Diisopropylether (70 mL) was added to the abovesolution to give a saturated solution. (The quantity of diisopropylether may vary based on the purity of the product.) The solution wasseeded with pure S_(P)-4 (>99%) and the mixture was gently stirred witha stir bar at room temperature for 20 h (formation of solid was observedafter 2 h). The solid was filtered, washed with 40 mL of the mixture ofdiisopropylether/dichloromethane (1:1) and dried to give white solid(16.6 g, 89.35% pure S_(P)-4 by NMR). This solid was suspended in 83 mLdichloromethane and refluxed for 3 h. The suspension was cooled to roomtemperature and stirred over night. The solid was filtered and washedwith 10 mL of cold DCM. The solid was dried under vacuum to give S_(P)-4(13.1 g, 99.48% pure by HPLC). 11 g of this solid was redissolved in 330mL of DCM under hot conditions. The solution was cooled to roomtemperature and left at this temperature over night. The crystallineproduct was filtered and dried to give 10.5 g of S_(P)-4 (99.74% byHPLC).

Compounds S_(P)-4 and R_(P)-4 may alternatively be prepared, inaccordance with the ninth or tenth embodiment, by reacting nucleoside(protected or unprotected) 3 with an isopropyl-alanyl-phosphoramidate(mixture of C and C′, C or C′), as shown in the following equation.

P. D. Howes et al. Nucleosides, Nucleotides & Nucleic Acids 2003, Vol.22, Nos. 5-8, pp. 687-689 (“Howes”) discloses 2′- and5′-phosphoramidates obtained by a reaction with t-butylmagnesiumchloride. There, Howes discloses that when a 3′-deoxy-cytidinenucleoside is reacted with(S)-2-[chloro-phenoxy-phosphorylamino]propionic acid methyl ester in thepresence of 1.2 equivalents of t-butylmagnesium chloride, selectivephosphorylation on the 2′-position occurred, but that with an additionalequivalent of t-butylmagnesium chloride selective phosphorylation on the5′-position occurred. This disclosure should be contrasted to that whichis disclosed in Howes' Scheme 1.

Example 5-1 Preparation of(S)-2-[(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester

To a stirred solution of 4-nitrophenyl phoshorodichloridate 12.8 g, 50mmol) in dichloromethane (100 mL) was added a solution of phenol andtriethylamine (7.7 mL, 55 mmol) in dichloromethane (100 mL) at −78° C.over a period of 20 min. The mixture was stirred at this temperature for30 min and then transferred to another round bottom flask containingL-alanine isopropyl ester hydrochloride (8.38 g, 50 mmol) indichloromethane (100 mL) at 0° C. To the mixture was added secondportion of triethylamine (14.6 mL, 105 mmol) over a period of 15 min.The mixture was stirred at 0° C. for 1 h and then the solvent wasevaporated. The residue was triturated with ethyl acetate (150 mL) andthe white solid was filtered off. The filtrate was concentrated underreduced pressure to give pale yellow oil. The crude compound waschromatographed using 0-20% ethyl acetate/hexanes gradient to giveproduct (17 g, 83% yield) as a mixture of diastereomers in about 1:1ratio. ³¹P NMR (162 MHz, DMSO-d6): δ −0.31, −0.47; ¹H NMR (400 MHz,DMSO-d6): δ 8.31-8.27 (m, 2H), 7.51-7.37 (m, 4H), 7.27-7.19 (m, 3H),6.70-6.63 (m, 1H), 4.85-4.78 (m, 1H), 3.97-3.86 (m, 1H), 1.21-1.19 (m,3H), 1.11-1.09 (m, 6H); MS (ESI) m/z 407 (M−1)⁺. ³¹P NMR (162 MHz,CDCl₃): δ −2.05, −2.10; ¹H NMR (400 MHz, CDCl₃): δ 8.22 (d, J=9.2 Hz,2H), 7.41-7.33 (m, 4H), 7.26-7.18 (m, 3H), 5.05-4.96 (m, 1H), 4.14-4.05(m, 1H), 3.93-3.88 (m, 1H), 1.38 (d, J=6.8 Hz, 3H), 1.22 (dd, J=6.2 &3.0 Hz, 6H); MS (ESI) m/z 407 (M−1)⁺.

Example 5-2 Preparation of S_(P)-4/R_(P)-4

To a stirred solution of1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione(130 mg, 0.5 mmol) in dry THF (1.5 mL) was added a 1.0M solution oftert-butylmagnesium chloride (1.05 mL, 1.05 mmol, 2.1 equiv)) at roomtemperature over a period of 5 min. After 30 min, a solution of(S)-2-[(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester (1:1 mixture of isomers, 408 mg, 1 mmol) in THF (1.5 mL)was added drop-wise over a period of 5 min. The mixture was allowed tostir at room temperature for 48 h and then quenched with saturatedaqueous NH₄Cl (20 mL). The mixture was partitioned between ethyl acetate(50 mL) and water (20 mL). The combined organic extract was dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give a pale yellow residue. Column chromatography of theresidue using 0-2% MeOH/dichloromethane gradient gave a white foamysolid (125 mg, 47% yield, mixture of S_(P)-4/R_(P)-4 in about 3.05:1.0ratio).

Example 6 Preparation and non-chromatographic isolation of(S)-2-[(S)-(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester

L-alanine isopropyl ester hydrochloride (330 g, 1.97 mol) was pre-driedby co-evaporation with toluene (2×400 mL) under reduced pressure andthen dried in a vacuum oven (50° C., 0.2 mmHg, 17 h). To a stirredsolution of 4-nitrophenyl phosphorodichloridate (500.0 g, 1.953 mol) inanhydrous dichloromethane (3.0 L) was added a solution of phenol (183.8g, 1.953 mol) and triethylamine (300 mL, 2.15 mol) in dichloromethane(900 mL) at −60° C. internal temperature over a period of 3 hours. Themixture was stirred at this temperature for additional 30 min and thenallowed to warm up to −5° C. over 2.5 hours. The pre-dried amino acidester was added at −5-0° C. under an atmosphere of nitrogen over 10mins. The residue of aminoester salt in the addition flask wastransferred to the reaction mixture via rinsing with dichloromethane(2×100 mL). The mixture was stirred at 0° C. for 40 mins and a secondportion of triethylamine (571 mL, 4.10 mol) was added over a period of40 mins at 0° C. The mixture was stirred at 0˜10° C. for 3 h and thenthe white solid (triethylamine hydrochloride) was filtered off andrinsed with dichloromethane (3×300 mL). The filtrate was concentratedunder reduced pressure and the residue was triturated with methylt-butyl ether (MTBE, 4 L). The additional solid salt thus formed wasfiltered off and rinsed with MTBE (3×150 mL). The filtrate wasconcentrated under reduced pressure to give clear light brown color oil.The residue was co-evaporated with hexanes (2×140 mL) to remove anyresidual MTBE and further dried under vacuum at 40° C. for 2 hours. Thedry residue was mixed with diisopropyl ether (IPE, 1.1 L) and stirred at5° C. in an ice-water bath. Small amount of crystal seeds of the desiredS_(P)-isomer of the product was added to the solution and the mixturewas stirred at 5° C. for over 22 h to form a medium thick slurry. Thiswas allowed to stand in a freezer (−10° C.) for 44 h. The precipitatedproduct was collected via filtration and rinsed with pre-cooled mixedsolvents of IPE and hexanes (1:1, 3×190 mL). The solid was dried undervacuum (0.5 mm Hg) at ambient temperature until a constant weight wasobtained to give 227.23 g (yield: 28.5%) as a white powder solid. Theratio of two diastereomers S_(P):Rp was 9.65/1 based on ³¹P NMR (162MHz, DMSO-d₆, δ −0.31 (S_(P)), −0.47). The product was recrystallized bydissolving in IPE (840 mL) while heating in a 60° C. bath. The abovesolution was stirred at room temperature for 1 h and then a small amountof crystal Sp isomer seeds was added. White powder solid was formedwithin 2 hours and the flask was stored in a freezer (−10° C.) for 16hours. A white and fine crystalline solid obtained was filtered, washedwith pre-cooled IPE (3×50 mL) and dried under vacuum (ambient, 0.5 mmHg) to a constant weight to give white fluffy solid (177.7 g, 22%overall yield or 44% overall yield based on theoretical yield of the Spisomer) with diastereomeric ratio of 48/1 based on P-NMR. Mp 62-66° C.

³¹P NMR (162 MHz, DMSO-d6): δ −0.31; ¹H NMR (400 MHz, DMSO-d6): δ8.30-8.27 (m, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.41-7.37 (m, 2H), 7.23-7.19(m, 3H), 6.66 (dd, J=13.6, 10.0 Hz, 1H), 4.86-4.78 (m, 1H), 3.97-3.86(m, 1H), 1.19 (d, J=7.2 Hz, 3H), 1.10 (d, J=6.4 Hz, 6H);

³¹P NMR (162 MHz, CDCl₃): δ −2.05; (162 MHz, DMSO-d6): δ −0.31; ¹H NMR(400 MHz, CDCl₃): δ 8.22 (d, J=9.2 Hz, 2H), 7.41-7.33 (m, 4H), 7.26-7.18(m, 3H), 5.05-4.96 (m, 1H), 4.14-4.05 (m, 1H), 3.93-3.88 (m, 1H), 1.38(d, J=6.8 Hz, 3H), 1.22 (dd, J=6.2 & 3.0 Hz, 6H); ¹H NMR (400 MHz,DMSO-d6): δ 8.30-8.27 (m, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.41-7.37 (m,2H), 7.23-7.19 (m, 3H), 6.66 (dd, J=13.6, 10.0 Hz, 1H), 4.86-4.78 (m,1H), 3.97-3.86 (m, 1H), 1.19 (d, J=7.2 Hz, 3H), 1.10 (d, J=6.4 Hz, 6H)

MS (ESI) m/z 407 (M−1)⁺.

The stereochemistry of 8 (S_(P)-isomer) has been confirmed by singlecrystal X-ray crystallography, see details provided below.

Example 7 Separation of the diastereomeric mixture(S)-2-[(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester by SFC

A sample of the mixture of diastereomers (4.8 g) enriched with theR_(P)-isomer was subjected to SFC using a ChiralPak AD-H (2×15 cm)column and eluted with 35% isopropanol in carbon dioxide at 100 bar. Aninjection loading of 4 mL of sample at a concentration of 17 mg/mL ofmethanol was used. The R_(P)-isomer[(S)-2-[(R)-(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester] eluted first. The appropriate fractions of the multipleruns were combined and concentrated under reduced pressure to give 2.9 gof the R_(P)-isomer[(S)-2-[(R)-(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester] as a light yellow viscous oil and 1.9 g of theS_(P)-isomer[(S)-2-[(S)-(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester] as a white solid. Analytical data of R_(P)-isomer issimilar to the product isolated by the above crystallization method.

Analytical Data for(S)-2-[(R)-(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester (8, R_(P)-isomer)

³¹P NMR (162 MHz, DMSO-d6): δ −0.47; ¹H NMR (400 MHz, DMSO-d6): δ8.30-8.27 (m, 2H), 7.46-7.38 (m, 4H), 7.27-7.20 (m, 3H), 6.68 (dd,J=13.8, 10.2 Hz, 1H), 4.86-4.77 (m, 1H), 3.97-3.86 (m, 1H), 1.20 (d,J=7.2 Hz, 3H), 1.10 (dd, J=6.2, 2.2 Hz, 6H); MS (ESI) m/z 407 (M−1)⁺.

Example 8-1 Preparation of racemic2-[(4-chloro-phenoxy)-phenoxy-phosphorylamino]propionic acid isopropylester (±)

To a stirred solution of 4-chloro-phenyl phoshorodichloridate (2.45 g,10.0 mmol) in dichloromethane (20 mL) was added a solution of phenol(0.94 g, 10 mmol) and triethylamine (1.56 mL, 11 mmol) indichloromethane (20 mL) at −78° C. over a period of 20 min. The mixturewas stirred at this temperature for 30 min and then transferred toanother round bottom flask containing L-alanine isopropyl esterhydrochloride (1.67 g, 10 mmol) in dichloromethane (50 mL) at 0° C. Tothe mixture was added second lot of triethylamine (2.92 mL, 21 mmol)over a period of 15 min. The mixture was stirred at 0° C. for 1 h andthen the solvent was evaporated. The residue was triturated with ethylacetate (30 mL) and the white solid was filtered off. The filtrate wasconcentrated under reduced pressure to give pale yellow oil. The crudecompound was chromatographed using 10-20% ethyl acetate/hexanes gradientto give product (2.0 g, 50% yield) as a mixture of diastereomers inabout 1:1 ratio. ³¹P NMR (162 MHz, CDCl₃): δ −1.58, −1.62; ¹H NMR (400MHz, CDCl₃): δ 7.06-7.51 (m, 8H), 7.15-7.28 (m, 2H), 7.29-7.47 (m, 2H),4.0-4.10 (m, 1H), 3.82-3.88 (m, 3H), 1.35-1.36 (dd, 6H); 1.19-1.22 (m,3H). MS (ESI) m/z 398 (M−1)⁺. The resultant product is purified byextraction, crystallization, or chromatography, as noted above.

Example 8-2 Preparation of (S)-Isopropyl2-((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2yl)methoxy)(phenoxy)-phosphorylamino)propanoate (4)

To a stirred solution of1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione(3, 2.6 g, 10 mmol) in dry THF (50 mL) was added a 1.7 M solution oftert-butylmagnesium chloride (12.4 mL, 21 mmol, 2.1 equiv)) at roomtemperature over a period of 15 min. After 30 min, a solution of racemic(2-[(4-chloro-phenoxy)-phenoxy-phosphorylamino]propionic acid isopropylester (4.08 g, 10 mmol) in THF (15 mL) was added drop wise over a periodof 10 min. The mixture was allowed to stir at room temperature for 72.TLC co-spot with authentic product showed around 5% of the desiredproduct had formed compared to the starting nucleoside.

Example 9-1 Preparation of racemic2-[(2-chloro-phenoxy)-phenoxy-phosphorylamino]propionic acid isopropylester (±)

To a stirred solution of 2-chloro-phenyl phoshorodichloridate (9.8 g, 40mmol) in dichloromethane (80 mL) was added a solution of phenol (3.76 g,40 mmol) and triethylamine (6.16 mL, 44 mmol) in dichloromethane (80 mL)at −78° C. over a period of 20 min. The mixture was stirred at thistemperature for 30 min and then transferred to another round bottomflask containing L-alanine isopropyl ester hydrochloride (6.7 g, 40mmol) in dichloromethane (150 mL) at 0° C. To the mixture was addedsecond portion of triethylamine (11.6 mL, 84 mmol) over a period of 15min. The mixture was stirred at 0° C. for 1 h and then the solvent wasevaporated. The residue was triturated with ethyl acetate (100 mL) andthe white solid was filtered off. The filtrate was concentrated underreduced pressure to give pale yellow oil. The crude compound waschromatographed using 10-20% ethyl acetate/hexanes gradient to giveproduct (11.3 g, 72% yield) as a mixture of diastereomers in about 1:1ratio. ³¹P NMR (162 MHz, CDCl₃): δ −1.58, −1.61; ¹H NMR (400 MHz,CDCl₃): δ 7.06-7.51 (m, 8H), 5.02-5.94 (m, 1H), 4.10-4.16 (m, 1H),3.31-3.94 (m, 1H), 1.18-1.35 (m, 3H), 1.38-1.40 (dd, 6H); MS (ESI) m/z398 (M−1)⁺. The resultant product is purified by extraction,crystallization, or chromatography, as noted above.

Example 9-2 Preparation of (S)-isopropyl2-((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2yl)methoxy)(phenoxy)-phosphorylamino)propanoate

To a stirred solution of1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione(3, 2.6 g, 10 mmol) in dry THF (50 mL) was added a 1.7 M solution oftert-butylmagnesium chloride (12.4 mL, 21 mmol, 2.1 equiv)) at roomtemperature over a period of 15 min. After 30 min, a solution of(2-[(2-chloro-phenoxy)-phenoxy-phosphorylamino]propionic acid isopropylester (racemic 4.08 g, 10 mmol) in THF (15 mL) was added drop wise overa period of 10 min. The mixture was allowed to stir at room temperaturefor 72 h. TLC co-spot with authentic product showed around 5-10% of thedesired product had formed compared to the starting nucleoside.

Example 10-1 Preparation of racemic2-[(2,3,4,5,6-pentafluoro-phenoxy)-phenoxy-phosphorylamino]propionicacid isopropyl ester (±)

To a stirred solution of pentafluorophenyl phoshorodichloridate (6.0 g,20 mmol) in dichloromethane (40 mL) was added a solution of phenol andtriethylamine (3.08 mL, 22 mmol) in dichloromethane (40 mL) at −78° C.over a period of 20 min. The mixture was stirred at this temperature for30 min and then transferred to another round bottom flask containingL-alanine isopropyl ester hydrochloride (3.35 g, 20 mmol) indichloromethane (100 mL) at 0° C. To the mixture was added second lot oftriethylamine (5.84 mL, 42 mmol) over a period of 15 min. The mixturewas stirred at 0° C. for 1 h and then the solvent was evaporated. Theresidue was triturated with ethyl acetate (60 mL) and the white solidwas filtered off. The filtrate was concentrated under reduced pressureto give pale yellow oil as a mixture of diastereomers in about 1:1ratio. ³¹P NMR (162 MHz, CDCl₃): δ −0.49, −0.58. The resultant productis purified by extraction, crystallization, or chromatography, as notedabove.

Example 10-2 Preparation of (S)-isopropyl2-((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2yl)methoxy)(phenoxy)-phosphorylamino)propanoate

To a stirred solution of1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione(3, 2.6 g, 10 mmol) in dry THF (50 mL) was added a 1.7M solution oftert-butylmagnesium chloride (12.4 mL, 21 mmol, 2.1 equiv)) at roomtemperature over a period of 15 min. After 30 min, a solution of cruderacemic (2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphorylamino]propionic acid isopropyl ester (4.08 g,10 mmol) in THF (15 mL) was added drop wise over a period of 10 min. Themixture was allowed to stir at room temperature for 72 h. TLC co-spotwith authentic product showed around 40-50% of the desired product hadformed compared to the starting nucleoside.

The preparation and purification of C or C′ provides for direct accessto either S_(P)-4 or R_(P)-4, as illustrated in the following examples.

Example 11 Preparation of S_(P)-4 (32 mg-scale)

To a stirred solution of1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione3 (32 mg, 0.12 mmol) in dry THF (1 mL) was added a 1M solution oftButylmagnesium chloride (0.26 mL, 0.26 mmol, 2.1 equiv)) at roomtemperature over a period of 3 min. After 30 min, a solution of(S)-2-[(S)-(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester (8, S_(P)-isomer) in THF (0.5 mL) was added drop wiseover a period of 3 min. The mixture was allowed to stir at roomtemperature for 42 h and then quenched with saturated aqueous NH₄Cl (10mL). The mixture was partitioned between ethyl acetate and water. Thecombined organic extract was dried over anhydrous sodium sulfate andconcentrated. The residue was chromatographed using 0-4%methanol/dichloromethane gradient to give S_(P)-4 as foamy solid (29 mg,44.5% yield). ¹H and ³¹P NMR agree to that which is disclosed herein.

Example 12 Preparation of S_(P)-4 (2.6 g-scale, without chromatography)

To a stirred solution of1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione(2.6 g, 10 mmol) in dry THF (50 mL) was added a 1.7 M solution oftert-butylmagnesium chloride (12.4 mL, 21 mmol, 2.1 equiv)) at roomtemperature over a period of 15 min. After 30 min, a solution of(S)-2-[(S)-(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester (8, Sp-isomer, 4.08 g, 10 mmol) in THF (15 mL) was addeddrop wise over a period of 10 min. The mixture was allowed to stir atroom temperature for 60 h and then quenched with saturated aqueous NH₄Cl(20 mL). The mixture was partitioned between ethyl acetate (150 mL) andsequentially, 10% aqueous Na₂CO₃ (3×20 mL) and water (20 mL). Thecombined organic extract was dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give a pale yellowresidue (3.8 g). The residue was dissolved in dichloromethane (7.6 mL)and then stirred for 20 h at room temperature. The white solid wasfiltered, washed with 1:1 IPE/dichloromethane (5 mL) and dried undervacuum to give pure product as white solid (1.85 g, 35% yield).

Example 13 Preparation of S_(P)-4 using NaHMDS

To a stirred solution of1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione(71 mg, 0.27 mmol) in dry THF (2.0 mL) was added a 2.0 M solution ofsodium bis(trimethylsilyl)amide (NaHMDS) in THF (270 μL, 0.54 mmol) at−78° C. over a period of 2 min. After 30 min, a solution of(S)-2-[(S)-(4-Nitro-phenoxy)-phenoxy-phosphorylamino]-propionic acidisopropyl ester (8, S_(P)-isomer, 111 mg, 0.27 mmol) in THF (1 mL) wasadded to the mixture. The reaction mixture was allowed stir at thistemperature for 2 h and then warmed to −20° C. at which temperature itwas stirred for additional 20 h. TLC indicated ˜30% of unreactednucleoside starting material. Hence, additional 0.5 equivalents of thereagent (55 mg, 0.14 mmol) in THF (0.5 mL) was added to the reactionmixture and stirred for another 6 h. The reaction mixture was quenchedwith saturated aqueous ammonium chloride solution and then partitionedbetween ethyl acetate and water. The combined organic extract was driedover anhydrous sodium sulfate and concentrated to give a light brownresidue. Column chromatography of the crude product using 0-5%methanol/dichloromethane gradient gave S_(P)-4 (22 mg, 15% yield),3′-phoshoramidate (5, S_(P)-isomer, 11.5 mg, 16% yield) and bisphosphoramidate (6, Sp, S_(P)-isomer, 12.6 mg).

Example 14 Preparation of R_(P)-4 (260 mg-scale)

To a stirred solution of1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione(260 mg, 1 mmol) in dry THF (6 mL) was added a 1.7 M solution oftert-butylmagnesium chloride (1.23 mL, 2.1 mmol, 2.1 equiv)) at roomtemperature over a period of 5 min. After 30 min, a solution of(S)-2-[(R)-(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester (8, R_(P)-isomer) in THF (3 mL) was added drop wise overa period of 3 min. The mixture was allowed to stir at room temperaturefor 96 h and then quenched with saturated aqueous NH₄Cl (10 mL). Themixture was partitioned between ethyl acetate (50 mL) and water (2×20mL). The combined organic extract was dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give a paleyellow residue (490 mg). The residue was chromatographed using 0-5%methanol/dichloromethane gradient to give product as a white solid (160mg, 30% yield).

The preparation of S_(P)-4 or R_(P)-4 may also be achieved by reacting3′-protected 3 with the appropriate reagent C or C′ or a mixturecontaining C and C′, as illustrated in the following examples.

Example 15 Preparation of S_(P)-4 with 3a as a Synthetic Intermediate

Example 15-1 Synthesis of5′-O-tert-Butyldimethylsilyl-2′-deoxy-2′-fluoro-2′-C-methyluridine (9)

To a stirred solution of 2′-deoxy-2′-fluoro-2′-C-methyluridine (3, 81.1g, 312 mmol) in dry pyridine (750 mL) was added drop-wise a solution ofTBDMSCl (103.19 g, 685.6 mmol) in dry pyridine (500 mL) over a period of45 min at ambient temperature. The reaction was allowed to stir atambient temperature for 24 h. Methanol (85 mL) was added to the reactionmixture and it was allowed to stir for 10 min and then the solvents weredistilled off under reduced pressure. Hot water (45° C.) (1 L) was addedto the reaction mass and the mixture extracted with ethyl acetate (2×500mL), washed with water (1×500 mL). The organic layer was dried overanhydrous sodium sulfate. Ethyl acetate was distilled off and theresidue obtained was co-evaporated with toluene (2×500 mL) to give crude9 as a white foam. Yield=116.9 g (quantitative). ¹H NMR (CDCl₃, 300MHz): δ 0.1 (s, 6H), 0.91 (s, 9H), 1.22 (d, 3H, J=21 Hz), 2.50 (s, 2H),3.75-4.05 (m, 4H), 5.54 (d, 1H, J=9 Hz), 5.73 (s, 1H), 6.0 (d, 1H, J=18Hz), 7.81 (d, 1H, J=9 Hz), 8.57 (br, s, 1H), 11.1 (s, 1H).

Example 15-2 Synthesis of5′-O-(tert-Butyldimethylsilyl)-3′-O-levulinyl-2′-deoxy-2′-fluoro2′-C-methyl-uridine (10)

To a stirred solution of nucleoside 9 (116.9 g, 312.1 mmol) in DCM (1 L)was added DMAP (30.5 g, 249.7 mmol) and this was allowed to stir at RTfor 20 min. A soln. of levulinic anhydride (133.6 g, 642.3 mmol) in DCM(200 mL) was added to the mixture and allowed to stir for 24 h. TLC ofthe mixture indicated completion of reaction. Cold water (500 mL) wasadded and the mixture stirred for 20 min. Layers were separated and theorganic layer was washed with sat. sodium bicarbonate solution (2×250mL), dried over anhydrous sodium sulfate and then the solvent wasdistilled under reduced pressure to give yellow oil. Crude yield: 197.6g (135%). The material was used as is for the next step. ¹H NMR (CDCl₃,300 MHz) δ 0.11 (s, 6H), 0.94 (s, 9H), 1.34 (d, 3H, J=21 Hz), 2.22 (s,3H), 2.6-2.89 (m, 4H), 3.72 (m, 1H), 4.01 (d, 1H, J=12 Hz), 4.23 (d, 1H,J=9 Hz), 5.33 (dd, 1H, J=15 Hz), 5.73 (d, 1H, J=6 Hz), 6.26 (d, 1H, J=15Hz), 8.12 (d, 1H, J=12 Hz), 8.72 (br, s, 1H).

Example 15-3 Synthesis of 3′-O-levulinyl-2′-deoxy-2′-fluoro2′-C-methyl-uridine (3a)

Crude 10 (197.6 g, 312.1 mmol) was dissolved in DCM (1 L) to which wasadded TEA.3HF (50.3 g, 312.1 mmol) and allowed to stir overnight atambient temperature. TLC of the mixture indicated about 50% completionof reaction. Another equivalent of TEA.3HF (50.3 g, 312.1 mmol) wasadded and the reaction mixture was allowed to stir for 6 h. TLC at thispoint indicated about 10% of unreacted starting material. Another 0.25eq of TEA.3HF (12.5 g, 78.0 mmol) was added and the reaction mixture wasallowed to stir overnight. Reaction mixture was concentrated to drynessto give yellow oil. Crude from all the batches was purified by columnchromatography on silica gel (0-2% MeOH in DCM) to give 124.1 g of3′-levulinate as a white foam solid (90% purified yield over three stepsfrom 2′-deoxy-2′-fluoro-2′-C-methyluridine). ¹H NMR: (CDCl₃, 400 MHz) δ1.55 (d, 3H, CH₃, J=20 Hz), 2.36 (s, 3H, CH3), 2.8-3.03 (m, 5H, CH2CH3),3.91-3.96 (dd, 1H, CH″), 4.2-4.25 (m, 1H, CH′), 4.34 (dd, 1H, CH, J=8Hz), 5.25 (dd, 1H, J=16 Hz), 5.93 (d, 1H, J=8 Hz), 8.20 (d, 1H, J=8 Hz),9.18 (s, 1H).

Example 15-4 Stereoselective synthesis of(S)-2-{[(1R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-4-(R)-fluoro-3-(4-oxopentanoyl)-4-methyl-tetrahydro-furan-2-ylmethoxy]-phenoxy-phosphorylamino}-propionicacid (S)-isopropyl ester (11)

To a solution of the nucleoside (3a, 1.00 mmol, 358 mg) in 5 mlanhydrous THF that was cooled to 0° C. was added tBuMgCl (1.7 M in THF,2 eq) and allowed it to warm to ambient temperature and stirred for halfhour. To this mixture was added the reagent (ca. 97% chiral purity)(S)-2-[(S)-(4-nitro-phenoxy)-phenoxy-phosphorylamino]propionic acidisopropyl ester (8, S_(P)-isomer) (408 mg, 1.00 mmol, 1.00 eq.) in onelot and allowed it to stir at rt. After 16 h, there was ˜30% startingmaterial left. The reaction mixture was quenched with saturated NH₄Clsolution 10 ml, and the aqueous phase was extracted with ethyl acetate(3×25 ml). The combined organic layer was washed with brine and driedover anhydrous sodium sulfate and evaporated to dryness to give a paleyellow foam (500 mg). This was purified by silica gel chromatographyusing 2-5% methanol in methylene chloride to give the product as a whitefoam (275 mg) of about 97% P chiral purity and unreacted startingmaterial (162 mg). Based on consumed starting material, the yield was76%. ³¹P NMR (CDCl₃, 162 MHz): 3.7 ppm; ¹H NMR (CDCl₃, 400 MHz): δ 1.22(dd, 6H, J=6.4 Hz), 1.37 (s, 3H), 1.58 (s, 3H), 2.18 (s, 3H), 2.63-2.9(m, 4H), 4.0 (d, 1H, J=8 Hz), 4.2-4.33 (m, 1H), 4.57 (d, 1H, J=8 Hz),4.96-5.00 (sept, 1H), 5.2 (dd, 1H, J=9 Hz), 5.42 (d, 1H, J=8 Hz), 6.19(d, 1H, J=18 Hz), 7.15-7.35 (m, 5H), 7.5 (d, 1H, J=5.6 Hz), 8.2 (br, s,1H).

Example 15-5 Synthesis of(S)-2-{[(1R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-4-(R)-fluoro-3-hydroxy-4-methyl-tetrahydro-furan-2-ylmethoxy]-phenoxy-phosphorylamino}-propionicacid (S)-isopropyl ester (S_(P)-4)

A solution of sodium sulfite was prepared by adding Na₂S₂O₃ (1.51 g) andNa₂S₂O₅ (0.57 g) in water (25 mL). To a solution of the levulinate (11,250 mg, 0.40 mmol) in anhydrous THF (2.5 mL) was added 1.0 ml of thesodium sulfite solution. This was allowed to stir at room temperaturefor 4 h. The reaction mixture was poured in to water (15 mL) andextracted with ethyl acetate (3×25 mL) dried and evaporated to givequantitatively a white solid product with about 97% P chiral puritywhich matched the physical and spectral properties of S_(P)-4 produceddirectly from the unprotected nucleoside.

Example 16 Alternative Procedure for Preparing S_(P)-4 from 3a

To a stirred solution of 4-oxo-pentanoic acid(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-4-fluoro-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester (3a, 210 mg, 0.59 mmol) in dry THF (1.5 mL) was added a 1.7 Msolution of tert-butylmagnesium chloride (1.07 mL, 1.82 mmol) at roomtemperature over a period of 2 min. Initially, a white precipitate wasobserved and after 10 min the reaction mixture turned to dark yellowsolution. After 30 min, a solution of(S)-2-[(S)-(4-nitrophenoxy)-phenoxy-phosphorylamino]-propionic acidisopropyl ester (8 (S_(P)-isomer), 382 mg, 0.94 mmol) in THF (1.5 mL)was added drop wise over a period of 3 min. The mixture was heated at40° C. for 5 h at which time TLC and ¹H NMR indicated less than 2% ofunreacted starting material. The reaction was quenched with saturatedaqueous ammonium chloride and then partitioned between ethyl acetate andwater. The combined organic layer was washed with 10% aqueous Na₂CO₃solution (3×10 mL), followed by water. The organic layer was dried overanhydrous sodium sulfate and concentrated to give brown color residue(410 mg). The crude product was dissolved in tetrahydrofuran (1.0 mL)and then was added an aqueous solution of the mixture of sodium sulfite(37 mg, 0.295 mmol) and sodium metabisulfite (224 mg, 1.18 mmol) in 1 mLof water. The mixture was heated at 45° C. for 20 h at which stage onlyabout 10% conversion was observed by TLC, hence the additional sodiumsulfite (74 mg) and sodium metabisulfite (448 mg) was added and theheating was continued for additional 52 h. At this time, about 40%conversion observed by TLC. The reaction mixture was partitioned betweenwater and ethyl acetate. The combined organic layer was dried overanhydrous sodium sulfate and concentrated to give a brown residue (210mg). Column chromatography of the residue using 0-5% MeOH/DCM gradientgave unreacted starting material (89 mg) and S_(P)-4 (57 mg, 18% yield,24% based on recovered starting material).

Example 17 Preparation of S_(P)-4 with 3c as a Synthetic Intermediate

Example 17-1 Preparation of1-[(2R,3R,4R,5R)-4-(tert-butyldimethylsilanyloxy)-3-fluoro-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl]-1H-pyrimidine-2,4-dione,12

To a solution of 3 (10.0 g, 38.43 mmol) in pyridine (50 mL) were addeddichloromethane (50 mL). The solution was cooled to 0° C. To thesolution was added 4,4′-dimethoxytrityl chloride (14.32 g, 42.27 mmol)and the solution was stirred at 0° C. for 5 h. Methanol (5 mL) was addedto quench the reaction. The solution was concentrated to dryness underreduced pressure and the residue was partitioned between ethyl acetate(500 mL) and water (50 mL). The organic solution was washed with brine(50 mL) and dried (sodium sulfate, 4 g). The solvent was removed underreduced pressure and the residue was dissolved in dichloromethane (100mL). To the solution were added imidazole (7.83 g, 115 mmol) andt-butyldimethylsilyl chloride (8.68 g, 57.6 mmol). The solution wasstirred at ambient temperature for 16 h. Methanol was added to quenchthe reaction (5 mL) and the solvent was removed under reduced pressureand the residue was partitioned between ethyl acetate (500 mL) and water(50 mL). The organic solution was dried (sodium sulfate, 4 g) andevaporated under reduced pressure. The residue was purified by columnchromatography (10-40% EtOAc in Hexane) to give 5′-O-DMT-3′-O-tBDMSintermediate product. This is turn was treated with 1% trifluoroaceticacid in dichloromethane (200 mL). The solution was stirred at ambienttemperature for 1 h. Water (20 mL) was added and the solution wasstirred at ambient for another 1 h. Methanol (5 mL) was slowly added andthe solution was stirred at ambient for another 1 h. Ammonium hydroxidewas added to adjust the solution pH to 7. The organic solution wasseparated, dried (sodium sulfate, 4 g) and evaporated to dryness underreduced pressure. The residue was purified by silica gel columnchromatography (1-5% methanol in dichloromethane) to give 12 as a whitesolid 7.5 g in 50% yield over the three steps. ¹H NMR (DMSO-d6) 6 (ppm)11.48 (br s, 1H, NH), 7.94 (d, 1H, H-6), 6.00 (d, 1H, H-1′), 5.69 (d,1H, H-5), 4.06 (dd, 1H, 3′-H), 3.85 (m, 2H, H-5′ a, H-4′), 3.58 (br d,1H, H-5′ b), 1.27 (d, 3H, 2-CH₃), 0.89 (s, 9H, C(CH₃)₃), 0.12 (s, 6H,Si(CH₃)₂).

Example 17-2 Preparation of S_(P)-4 using1-[(2R,3R,4R,5R)-4-(tert-butyldimethylsilanyloxy)-3-fluoro-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl]-1H-pyrimidine-2,4-dione(3c)

To a stirred solution of1-[(2R,3R,4R,5R)-4-(tert-butyldimethylsilanyloxy)-3-fluoro-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl]-1H-pyrimidine-2,4-dione(12, 374 mg, 1 mmol) in dry THF (3 mL) was added a 1.7 M solution oftert-butylmagnesium chloride (1.8 mL, 3.1 mmol)) at room temperatureover a period of 2 min. Initially, a white precipitate was observed andafter 10 min the reaction mixture turned to clear dark yellow solution.After 30 min, a solution of(S)-2-[(S)-(4-nitrophenoxy)-phenoxy-phosphorylamino]-propionic acidisopropyl ester (8, S_(P)-isomer, 653 mg, 1.6 mmol) in THF (2.5 mL) wasadded drop wise over a period of 3 min. The mixture was heated at 40° C.for 20 h at which time TLC and ¹H NMR indicated less than 5% ofunreacted starting material. The reaction mixture was quenched withsaturated aqueous ammonium chloride and then partitioned between ethylacetate and water. The organic layer was washed with 10% aqueous Na₂CO₃solution (3×10 mL), followed by water (20 mL). The organic layer wasdried over anhydrous sodium sulfate and concentrated to give brownresidue containing 3c (850 mg). The crude product was dissolved intetrahydrofuran (2 mL) and was added 0.8 mL of 80% aqueous formic acidat room temperature. The reaction mixture was heated at 50° C. for 96 h.About 70% conversion was observed by TLC. The reaction mixture waspoured into cold saturated aqueous sodium bicarbonate and thenpartitioned between ethyl acetate and water. The combined organic layerwas dried over anhydrous sodium sulfate and concentrated to give brownresidue (220 mg). Column chromatography of the residue using 0-5%MeOH/DCM gradient gave unreacted starting material (21 mg) and S_(P)-4(77 mg, 35% yield, 39% yield based on recovered starting material).

Example 18 Preparation of S_(P)-4 with 3d as a Synthetic Intermediate

Example 18-1 Preparation of 3d

To a stirred solution of 3 in pyridine (20 mL) at 0° C. was addedTIPDS-Cl drop-wise over a period of 15 min. The mixture was slowlyallowed to warm to room temperature at which temperature it was stirredfor 16 h. The pyridine was evaporated and the residue was co-evaporatedwith toluene (50 mL). The residue was then triturated with hexanes andthe white precipitate was filtered off using a pad of Celite. Thefiltrate was concentrated under reduced pressure to give a foamy solid(12.97 g). The crude product (13) was redissolved in tetrahydrofuran (75mL) and was added an aqueous solution of TFA (75 mL, 1:1 TFA/water) at0° C. over a period of 20 min. The mixture was stirred at thistemperature for 6 h. TLC indicated ˜5% of starting material. Thereaction mixture was quenched with saturated aqueous NaHCO₃ until pH 8and then extracted with ethyl acetate. The combined organic extract waswashed with water, dried and concentrated to give white crystallinesolid. Further trituration of the solid with hexanes (30 mL) gave whitesolid which was filtered and dried under high vacuum to give 3d (10.1 g,84% yield over 2 steps). ¹H NMR (400 MHz, CDCl₃): δ 8.83 (bs, 1H), 7.94(bd, J=6.0 Hz, 1H), 6.10 (bd, J=18.4 Hz, 1H), 5.71 (d, J=8.2 Hz, 1H),4.43 (bs, 1H), 4.36 (dd, J=22.6, 9.0 Hz, 1H), 4.27 (bs, 1H), 4.10 (d,J=13.2 Hz, 1H), 4.03 (d, J=9.2 Hz, 1H), 3.92 (d, J=13.2 Hz, 1H), 1.39(d, J=22.0 Hz, 3H), 1.11-0.92 (m, 28H).

Example 18-2 Preparation of S_(P)-4

To a stirred solution of 3d (520 mg, 1 mmol) in dry THF (5 mL) was addeda 1.7M solution of tert-butylmagnesium chloride (1.8 mL, 3.1 mmol, 3.1equiv)) at room temperature over a period of 15 min. After 30 min, asolution of(S)-2-[(S)-(4-nitro-phenoxy)-phenoxyphosphorylamino]propionic acidisopropyl ester (8, S_(P)-isomer, 653 mg, 1.6 mmol) in THF (1 mL) wasadded drop wise over a period of 3 min. The mixture was allowed to stirat room temperature for 60 h. ¹H and ³¹P NMR of the crude sampleindicated mixture of diastereomers in about 1:0.76. The reaction mixturewas quenched with saturated aqueous NH₄Cl (20 mL). The mixture waspartitioned between ethyl acetate (150 mL) and sequentially, 10% aqueousNa₂CO₃ (3×20 mL) and water (20 mL). The combined organic extract wasdried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to give a pale yellow residue (14, 878 mg). The abovecompound, 14, was redissolved in tetrahydrofuran (3 mL) and then wasadded 80% aqueous formic acid. The mixture was heated at 55° C. for 20h. The reaction mixture was cooled to 0° C., and then quenched withsaturated aqueous sodium bicarbonate (pH 7.0). The reaction mixture wasthen partitioned between ethyl acetate and water. The combined organiclayer was dried over sodium sulfate and concentrated to give 560 mg ofthe residue. The residue was chromatographed using 0-5%methanol/dichloromethane gradient to give unreacted starting material(14, 242 mg) and S_(P)-4 (80 mg, 15% yield) as a white solid.

Example 19 Preparation of Isotopically Labeled S_(P)-4

Example 19-1 Preparation of1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)pyrimidine-2,4(1H,3H)-dione, 16

Uridine (15, 100.0 g, 409.5 mmol) was co-evaporated to dryness withanhydrous pyridine (600 mL) and re-suspended in anhydrous pyridine (700mL). To this stirred fine suspension was added1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (135.7 g, 482.5 mmol) over60 min at ambient temperature. After stirring the fine suspension for 17h at ambient temperature, the reaction was quenched by adding methanol(20 mL) and then concentrated under reduced pressure. The residue waspartitioned between ethyl acetate (1.5 L) and water (2 L). The organiclayer was further washed with 5% hydrochloric acid (2×1 L), brine (500mL), dried over solid sodium sulfate (50 g), filtered and concentratedunder reduced pressure to the crude product, ca 250 g. The residue wassubjected to a filtration column using silica gel (1.75 kg) and agradient of ethyl acetate in hexanes 20-65%. The pure product fractionsas judged by a homogenous TLC (Rf 0.55 in 1:1 hexanes-ethyl acetate)were combined and concentrated under reduced pressure and dried (40° C.,0.2 mm Hg, 24 h) to afford 145.5 g (76%) of 16 as a white foam solid. Anadditional fraction (35 g) of slightly impure 16 was also collected. ¹HNMR (DMSO-d₆) δ (ppm) 11.35 (s, 1H, NH), 7.66 (d, 1H, J=7.6 Hz, H-6),5.57 (d, 1H, J=4.8 Hz, 2′-OH), 5.50-5.49 (m, 2H, 1′-H and H-5),4.14-4.18 (m, 3H, 2′, 3′, 4′-H), 3.97-3.87 (m, 2H, 5′-Ha and Hb),1.02-0.95 (m, 28H, CH(CH₃)₂).

Example 19-2 Preparation of1-((6aR,8R,9aR)-2,2,4,4-tetraisopropyl-9-oxotetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)pyrimidine-2,4(1H,3H)-dione, 17

To a dry three-necked round flask were added anhydrous DCM (600 mL) andDMSO (30.82 g, 394.5 mmol). The solution was cooled to −78° C. in a dryice/acetone bath under an atmosphere of nitrogen. Trifluoroaceticanhydride (neat, 77.7 g, 369.8 mmol) was added via a syringe over 40mins and afforded a cloudy mixture. To the mixture a solution of uridinederivative 16 in DCM (600 mL) was added dropwise over 75 mins at −78° C.via an addition funnel. The heterogeneous mixture was stirred for 2 h at−78˜−65° C. and then anhydrous triethylamine (92 mL) was added via asyringe quickly to form a clear light yellow solution. After 1 h at lowtemperature, the reaction was complete as shown by TLC (30% EtOAc inhexanes). The cooling bath was removed and the reaction mixture waswarmed up slowly to ambient temperature over 1 h. The reaction wasquenched by addition of sat. NH₄Cl (180 mL). Water (200 mL) was addedand organic layer was separated. The aqueous layer was extracted againwith DCM (300 mL). The combined organic layer was washed with water(3×400 mL), brine (150 mL), and dried over Na₂SO₄. Removal of solventafforded a sticky brown residue.

The crude oil residue (contained trace of DCM) was stored overnight inthe freezer. After overnight, some crystal solid was observed in theoil. The oil was dissolved in 500 ml hexanes at ambient temperature. Thesolution was stored in the freezer for 24 hours and more solid wasformed. Solid was collected via filtration and rinsed with cold 10% DCMin hexanes (1 L) to remove most of the orange color. The solid (17) wasdried under vacuum for 2 h and then air dried for 24 h. The solidweighed 21 g after dried at 50° C. under vacuum. The filtrate wasconcentrated and the residue was purified via column chromatography(10-70% ethyl acetate in hexanes) to afford an additional 37 g (combinedyield of 97%) of 17 as a light orange solid.

Example 19-3 Preparation of1-((2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-3-¹³C-perdeuteriomethyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione,18

Magnesium (3.53 g, 147 mmol), washed with 5% aqueous hydrochloric acidand dried (50° C., 0.2 mm Hg, 24 h), was put into a two neck roundbottomed flask equipped with a magnetic stirrer and a condensor. Theflask was filled with argon gas and then anhydrous ether (80 mL) wasadded. To the magnesium in ether was added slowly perdeuterio-¹³C methyliodide (15.06 g, 110.3 mmol), which generated an exothermic reaction.After the reaction mixture was cooled down, the supernatant wastransferred to a solution of dried compound 17 (50° C., 0.2 mm Hg, 15 h)(10.0 g, 20.63 mmol) in anhydrous THF (1 L) at −50° C. over 20 min. Thetemperature was allowed to rise to −40° C. and the mixture was stirredat between −40 to −25° C. for 4 h. Upon completion of reaction, themixture was diluted with EtOAc (1 L) at −50° C. and then brine (300 mL)was added slowly. The organic layer was separated and then washed withsat'd ammonium chloride solution (300 mL×2) and dried with sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was dissolved in MeOH (250 mL). Ammonium fluoride (12 g) andTBAF (400 mg) were added. The resulting mixture was stirred at 90° C.for 7 h and then concentrated with silica gel (20 g) under reducedpressure. After thorough vacuum drying, the obtained residue waspurified by flash silica gel column chromatography (MeOH:CH₂Cl₂=1:20 to1:10) give compound 18 (5 g, 46%) as a white solid. ¹H NMR (DMSO-d₆) δ(ppm) 11.26 (s, 1H, NH), 7.65 (d, 1H, J=8.4 Hz, H-6), 5.77 (d, 1H, J=2.4Hz, H-1′), 5.57 (d, 1H, J=8.0 Hz, H-5), 5.46 (d, 1H, J=5.2 Hz, HO-3′),5.24 (d, 1H, J=2.4 Hz, HO-2′), 5.14 (t, 1H, J=5.6 Hz, HO-5′), 3.74-3.56(m, 4H, H-3′, 4′, 5′, 5″).

Example 19-4 Preparation ofO₂R,3R,4S,5R)-3-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-4-¹³C-perdeuteriomethyltetrahydrofuran-2-yl)methylacetate, 19

To a solution of compound 18 (5.00 g, 19.1 mmol) in anhydrous pyridine(100 mL) was added acetic anhydride (3 mL) at ambient temperature. Theresulting mixture was stirred at ambient temperature for 15 h, dilutedwith EtOAc (250 mL), washed with water (50 mL×3), and dried with sodiumsulfate. After filtration and concentration, the residue was purified byflash column chromatography (MeOH 0 to 5% in CH₂Cl₂) to give compound 19(4.0 g, 68%) as a gray solid.

Example 19-5 Preparation ofO₂R,3R,4R,5R)-3-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-4-¹³C-perdeuteriomethyltetrahydrofuran-2-yl)methylacetate, 20

To a solution of compound 19 (2.33 g, 6.73 mmol) in anhydrous CH₂Cl₂ (60mL) was added DAST (1.33 mL, 10.1 mmol) at −78° C. slowly. The resultingmixture was stirred for 30 min after exposed to ambient temperature. Anadditional two 2.33 g scale reactions and one 1.00 g scale reaction wereconducted exactly the same way. All four reaction mixtures werecombined, diluted with CH₂Cl₂ (300 mL), and washed with ice-water (100mL×2) and then cold aqueous NaHCO₃ solution (100 mL×2). After drying,filtration, and concentration, the residue was purified by flash silicagel column chromatography (EtOAc 0% to 50% in hexanes, compound came outat around 48%) to give compound 20 (2.0 g, 24% from total 7.99 g ofcompound 19) as a white solid. ¹H NMR (CDCl₃) δ (ppm) 8.27 (s, 1H, NH),7.55 (d, 1H, J=8.4 Hz, H-6), 6.17 (d, 1H, J=18.8 Hz, H-1′), 5.78 (dd,1H, J=1.2, 8.4 Hz, H-5), 5.12 (dd, 1H, J=9.6, 21.6 Hz, H-3′), 4.40-4.31(m, 3H, H-4′, 5′, 5″), 2.19 (s, 3H, CH₃), 2.15 (s, 3H, CH₃).

Example 19-6 Preparation of1-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-¹³C-perdeuteriomethyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione,21

To a solution of compound 20 (2 g, 5.74 mmol) in methanol (20 mL) wasadded n-butylamine (6 mL). The resulting mixture was stirred at rt for15 h and concentrated with silica gel in vacuo. The obtained residue waspurified by flash silica gel column chromatography (MeOH 0 to 10% inCH₂Cl₂) to give compound 21 (1.3 g, 85%) as a white solid. ¹H NMR(CD₃OD) 6 (ppm) 8.08 (d, 1H, J=8.0 Hz, H-6), 6.13 (d, 1H, J=18.4 Hz,H-1′), 5.70 (d, 1H, J=8.0 Hz, H-5), 3.99 (d, 1H, J=13.6 Hz, H-5′),3.97-3.91 (m, 2H, H-3′, 4′), 3.80 (dd, 1H, J=2.0, 12.8 Hz, H-5″), ESMS(M+1) estimated 265, observed 265.

Example 19-7 Preparation of (S)-Isopropyl2-((((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-¹³C-perdeuteriomethyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate,22

To a solution of the unprotected nucleoside 21 (207 mg, 0.783 mmol) andN-methylimidazole (0.4 ml, 5 mmol) in THF (4 mL) was added the pre-madephosphorochloridate in THF (1.0 M, 2.35 ml, 2.35 mmol) at 0° C.dropwise. The reaction was slowly warmed to ambient temperature over 1 hand then water (1 mL) and EtOAc (5 mL) were added. The organic solutionwas washed with sat. aq. mono basic sodium citrate (2×2 ml), sat. aq.NaHCO₃ (1×2 ml), dried (MgSO₄) and concentrated under reduced pressure.The crude was purified by silica column chromatography using 0 to 5%^(i)PrOH in CH₂Cl₂ as eluents to give the phosphoramidate, 22 (216 mg,52%, 1:1 mixture of P-diastereomers) as a white solid: ¹H NMR (400 MHz,DMSO-d₆) δ 11.54 (s, 1H), 7.56 (d, J=6.8 Hz, 1H), 7.40-7.35 (m, 2H),7.23-7.18 (m, 3H), 6.14-5.96 (m, 2H), 5.89 (dd, J=5.6, 25.6 Hz, 1H),5.55 (t, J=8.4 Hz, 1H), 4.85 (dq, J=1.6, 6.0 Hz, 1H), 4.44-4.32 (m, 1H),4.25 (m, 1H), 4.06-3.98 (m, 1H), 3.86-3.70 (m, 2H), 1.30-1.08 (m, 9H);³¹P NMR (162 MHz, DMSO-d₆) δ 4.90, 4.77; LRMS (ESI) [M+H]⁺ calculatedfor C₂₁ ¹³CH₂₇D₃FN₃O₉P 534.5. found 534.4.

Example 19-8 Preparation of(2S)-2-(((((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-¹³C-perdeuteriomethyltetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)amino)propanoicacid, 23

Phosphoramidate 22 (147 mg, 0.276 mmol) was suspended in triethylamine(2 mL) and water (0.5 mL), and heated at 60° C. for 30 h. Then thevolatile components were evaporated under reduced pressure. The crudewas purified by silica column chromatography by eluting with 50-70%¹PrOHin CH₂Cl₂ and then, 0 to 20% NH₄OH in ^(i)PrOH to give 23 as a whitesolid (95 mg, 83%): ¹H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J=8.4 Hz, 1H),5.98 (d, J=19.2 Hz, 1H), 5.52 (d, J=8.4 Hz, 1H), 4.02-3.81 (m, 4H), 1.10(d, J=6.8 Hz, 3H); ³¹P NMR (162 MHz, DMSO-d6) δ 8.12; LRMS (ESI) [M+H]⁺calculated for C₁₂ ¹³CH₁₇D₃FN₃O₉P 416.3. found 416.4.

Properties of Samples of R_(P)-4, 4, and S_(P)-4

Samples of R_(P)-4, 4, and S_(P)-4 were analyzed by X-Ray PowderDiffraction (XRPD), Nuclear Magnetic Resonance (NMR) spectrometry,Fourier Transform Infrared (FT-IR) spectroscopy, Differential Scanningcalorimetry (DSC), Thermal Gravimetric Analysis (TGA), Gravimetric VaporSorption (GVS), Thermodynamic Aqueous Solubility, and High PerformanceLiquid Chromatography (HPLC).

Example 20 X-Ray Powder Diffraction

Samples of R_(P)-4, 4, and S_(P)-4 were analyzed by X-Ray PowderDiffraction (XRPD) under the following regimen.

a. Bruker AXS/Siemens D5000

X-Ray Powder Diffraction patterns were collected on a Siemens D5000diffractometer using Cu Kα radiation (40 kV, 40 mA), θ-θ goniometer,divergence of V20 and receiving slits, a graphite secondarymonochromator and a scintillation counter. The instrument is performancechecked using a certified Corundum standard (NIST 1976). The softwareused for data collection was Diffrac Plus XRPD Commander v2.3.1 and thedata were analyzed and presented using Diffrac Plus EVA v 11.0.0.2 or v13.0.0.2.

Ambient Conditions

Samples run under ambient conditions were prepared as flat platespecimens using powder as received. Approximately 35 mg of the samplewas gently packed into a cavity cut into polished, zero-background (510)silicon wafer. The sample was rotated in its own plane during analysis.The details of the data collection are: angular range: 2 to 42° 2θ; stepsize: 0.05° 2θ; and collection time: 4 s.step⁻¹.

b. Bruker AXS C2 GADDS

X-Ray Powder Diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZstage, laser video microscope for auto-sample positioning and a HiStar2-dimensional area detector. X-ray optics consists of a single Gaelmultilayer mirror coupled with a pinhole collimator of 0.3 mm.

The beam divergence, i.e. the effective size of the X-ray beam on thesample, was approximately 4 mm. A θ-θ continuous scan mode was employedwith a sample—detector distance of 20 cm which gives an effective 20range of 3.2°-29.7°. Typically the sample would be exposed to the X-raybeam for 120 seconds. The software used for data collection was GADDSfor WNT 4.1.16 and the data were analyzed and presented using DiffracPlus EVA v 9.0.0.2 or v 13.0.0.2.

Ambient Conditions

Samples run under ambient conditions were prepared as flat platespecimens using powder as received without grinding. Approximately 1-2mg of the sample was lightly pressed on a glass slide to obtain a flatsurface.

X-Ray Powder Diffraction (XRPD)

4 was found by XRPD to be amorphous (see FIG. 1). High resolution XRPDanalysis of R_(P)-4 prepared according to Example 3 confirmed acrystalline solid exhibiting a different powder pattern to that ofS_(P)-4 (prepared according to Example 4, Method 4), which was alsoconfirmed to be a crystalline solid. The XRPD results table for R_(P)-4and S_(P)-4 are shown in Table 1 with all peaks exhibiting an intensityof ≦5% (R_(P)-4) and ≦3% (S_(P)-4) excluded.

TABLE 1 XRPD Data for R_(P)-4 and S_(P)-4. XRPD data for R_(P)-4 XRPDdata for S_(P)-4(Form 1) Angle Angle 2-Theta ° Intensity % 2-Theta °Intensity % 6.616 51.1 4.900 6.8 7.106 40.5 5.190 19.8 8.980 30.0 7.501100.0 11.020 21.7 8.355 4.1 11.559 77.1 8.965 7.7 11.950 12.8 9.619 21.213.023 5.2 10.145 3.6 14.099 6.2 14.393 4.9 15.121 5.7 16.300 7.0 15.6245.4 16.688 10.6 16.003 17.8 17.408 5.5 17.882 100.0 17.820 8.2 18.5678.8 18.262 31.5 19.564 22.7 18.600 6.3 20.280 5.6 18.900 7.3 20.728 42.519.150 6.1 21.047 19.9 19.696 4.8 21.671 22.0 20.398 4.4 21.943 23.320.710 6.9 22.214 18.9 21.950 6.1 23.074 28.5 22.175 12.2 24.145 30.322.511 5.6 24.355 39.1 22.924 3.1 25.366 7.6 23.360 6.5 26.146 36.223.538 7.1 27.000 9.0 23.910 7.4 27.313 15.6 24.873 3.7 27.677 22.725.123 4.9 28.219 12.8 25.649 4.2 28.661 6.2 26.748 5.2 29.450 6.827.339 3.7 29.735 9.4 27.646 3.5 31.372 8.2 28.066 3.1 31.967 10.929.050 3.0 32.295 6.4 29.541 3.6 33.001 11.4 30.178 3.8 33.774 11.831.648 3.1 34.385 6.6 32.721 3.5 34.734 6.5 33.154 3.0 35.600 7.3 33.9233.5 35.965 13.1 34.341 3.1 36.409 14.7 35.465 3.5 36.880 7.0 36.923 3.137.509 5.9 37.760 3.4 37.870 6.0 38.404 3.3 38.313 5.8 40.416 3.1 38.9438.4 40.093 6.6 40.511 7.8 41.429 6.5

A sample of S_(P)-4 was ground with a pestle and mortar, and thensuccessively passed through 500 and 250 μm sieves to yield the sample asa fine powder. This sample was reanalyzed by high resolution XRPD,confirming no form change had occurred.

Example 21 Crystallization Studies for S_(P)-4

Crystalline S_(P)-4 exhibits polymorphism. Thus, an aspect is directedto crystalline S_(P)-4 and its individual polymorphic forms. S_(P)-4 canexist in at least five polymorphic forms, designated as Forms 1-5.Furthermore, amorphous S_(P)-4 can also be prepared. A typicalcrystallization provides for dissolving about 100 mg of S_(P)-4 in anappropriate volume of crystallization solvent (acetonitrile (5 vol),chloroform (5 vol), n-butyl acetate (7 vol), dichloromethane (50 vol),anisole (7 vol), and 1:1 MTBE/heptane (50 vol)) and then allowing forevaporation of the solution at 5° C. Various crystalline forms wereobtained, but each form, upon filtration and/or drying, afforded Form 1.

Forms 1, 2 and 3 are a non-solvated form, 1:1 DCM solvate and 1:1chloroform solvate, respectively, as was confirmed by singe crystalX-ray and XRPD analysis. Forms 4 and 5 were obtained fromcrystallization of S_(P)-4 from solutions of acetonitrile and anisole,respectively. Sufficient data could not be collected to determinewhether Forms 4 and 5 are unsolvated, hydrated or solvated since singlecrystals of sufficient quality were not obtained. Forms 4 and 5transform to Form 1 on filtration. Two additional crystalline forms areobtained upon crystallization of S_(P)-4 from n-butyl acetate (^(n)BuAc)and a solution containing methyl-^(t)butyl ether (MTBE) and heptane;upon filtration both of these crystalline forms convert to Form 1. Forms2 and 3 also transform to Form 1 on isolation. Form 1 is a non-solvatedform that exhibits a broad melting endotherm with an onset temperatureof 94.3° C. and ΔH_(fus) of 24.0 kJ mol⁻¹. An additional XRPD pattern ofS_(P)-4 Form 1 is depicted in FIG. 4.

Example 21-1 S_(P)-4 Form 1

A peak listing of S_(P)-4 Form 1 is presented in Table 2.

Angle Intensity % 2-Theta ° % 5.0 74.8 7.3 100.0 7.8 2.2 8.2 6.8 8.8 9.39.4 23.5 10.0 8.4 11.4 4.2 13.3 3.0 14.2 6.1 14.9 3.5 16.1 7.9 16.6 13.217.3 15.4 17.7 10.1 18.1 42.6 18.4 7.6 18.7 11.4 18.9 5.7 19.3 5.0 19.62.9 20.2 8.5 20.5 11.5 20.8 3.6 21.8 7.2 22.0 14.5 22.4 9.6 23.2 5.323.4 5.8 23.5 4.6 23.8 7.4 24.0 3.1 24.7 2.5 25.0 13.0 25.5 3.1 26.6 4.527.2 3.2 27.5 2.2 28.1 2.9 30.0 3.2

Example 21-2 S_(P)-4 Form 2

An XRPD pattern of S_(P)-4 Form 2 is depicted in FIG. 5.

A peak listing of S_(P)-4 Form 2 is presented in Table 3.

Angle Intensity % 2-Theta ° % 4.9 44.1 5.1 19.1 6.9 62.1 8.7 6.8 9.828.6 10.1 10.4 13.7 7.0 16.7 3.1 19.5 8.9 19.8 45.5 20.1 18.6 20.4 3.620.6 25.6 20.9 15.9 21.1 10.9 22.1 3.4 24.6 38.7 24.7 100.0 25.1 61.226.1 53.3 39.0 6.3

Example 21-3 S_(P)-4 Form 3

An XRPD pattern of S_(P)-4 Form 3 is depicted in FIG. 6.

A peak listing of S_(P)-4 Form 3 is presented in Table 4.

Angle Intensity % 2-Theta ° % 5.0 10.0 6.9 23.3 9.8 22.6 19.7 34.8 20.6100.0 21.8 10.5 24.6 65.3 34.7 4.1

Example 21-4 S_(P)-5 Form 4

An XRPD pattern of S_(P)-4 Form 4 is depicted in FIG. 7.

A peak listing of S_(P)-4 Form 4 is presented in Table 5.

Angle Intensity % 2-Theta ° % 5.0 29.8 6.8 100.0 8.2 4.8 8.7 5.2 9.9 3.813.7 1.7 14.9 4.8 19.9 22.5 20.4 2.1 20.6 20.0 20.9 20.0 24.7 3.4 24.929.9 25.1 1.5 36.8 3.1

Example 21-5 S_(P)-4 Form 5

An XRPD pattern of S_(P)-4 Form 5 is depicted in FIG. 8.

A peak listing of S_(P)-4 Form 5 is presented in Table 6.

Angle Intensity % 2-Theta ° % 5.2 52.9 6.6 100.0 7.1 25.9 9.7 12.1 10.416.4 13.4 11.4 15.7 25.8 19.1 31.1 19.9 12.9 20.0 9.0 21.3 3.5 25.0 22.325.6 2.3 26.3 5.9 26.9 2.0 31.7 2.1

Example 21-6 S_(P)-4 (Amorphous)

An XRPD pattern for amorphous S_(P)-4 is depicted in FIG. 9.

Example 22 Single Crystal X-Ray Crystallography of S_(P)-4 and itsSolvates Example 22-1 Single Crystal X-Ray Crystallography of S_(P)-4(Form 1)

FIG. 10 shows an X-ray crystal structure for S_(P)-4 Form 1. There, thefigure shows a view of molecules of Form 1 from the crystal structureshowing the numbering scheme employed. Anisotropic atomic displacementellipsoids for the non-hydrogen atoms are shown at the 50% probabilitylevel. Hydrogen atoms are displayed with an arbitrarily small radius.

The structure solution was obtained by direct methods, full-matrixleast-squares refinement on F² with weighting w⁻¹=σ²(F_(o)²)+(0.0592P)²+(0.6950P), where P=(F_(o) ²+2F_(c) ²)/3, anisotropicdisplacement parameters, empirical absorption correction using sphericalharmonics, implemented in SCALE3 ABSPACK scaling algorithm. FinalwR²={Σ[w(F_(o) ²−F_(c) ²)²]/Σ[w(F_(o) ²)²]^(1/2)}=0.0871 for all data,conventional R₁=0.0329 on F values of 7090 reflections withF_(o)>4σ(F_(o)), S=1.016 for all data and 870 parameters. Final Δ/σ(max)0.001, Δ/σ(mean), 0.000. Final difference map between +0.534 and −0.36 eÅ⁻³.

TABLE 7 Single Crystal Parameters of Form 1 Molecular formulaC₂₂H₂₉F₁N₃O₉P₁ Molecular weight  529.45 Crystal system Monoclinic Spacegroup P2₁ a 20.0898(5) Å, α 90°, b  6.10290(10) Å, β 112.290(3)°, c23.0138(6) Å, γ 90° V 2610.79(10) Å³ Z   4 D_(c) 1.347 g · cm⁻¹ μ 1.475mm⁻¹ Source, λ Cu Kα, 1.54178 Å F(000)  1112 T 100(1) K CrystalColorless plate, 0.12 × 0.09 × 0.03 mm Data truncated to 0.80 Å θ_(max)74.48° Completeness 99.4% Reflections 14854 Unique reflections  7513R_(int)   0.0217

Example 22-2 Single Crystal X-Ray Crystallography of S_(P)-4 (Form 2)

FIG. 11 shows an X-ray crystal structure for S_(P)-4 Form 2. There, thisfigure shows a view of molecules of Form 2 from the crystal structureshowing the numbering scheme employed. The heteroatoms were resolvedisotropically due to very weak data. Hydrogen atoms are not displayed.

The structure solution was obtained by direct methods, full-matrixleast-squares refinement on F² with weighting w⁻¹=σ²(F_(o)²)+(0.0975P)²+(10.6969P), where P=(F_(o) ²+2F_(c) ²)/3, anisotropicdisplacement parameters, empirical absorption correction using sphericalharmonics, implemented in SCALE3 ABSPACK scaling algorithm. FinalwR²={Σ[w(F_(o) ²−F_(c) ²)²]/Σ[w(F_(o) ²)²]^(1/2)}=0.1883 for all data,conventional R₁=0.0741 on F values of 2525 reflections withF_(o)>4σ(F_(o)), S=1.05 for all data and 158 parameters. Final Δ/σ(max)0.000, Δ/σ(mean), 0.000. Final difference map between +1.388 and −0.967e Å⁻³.

TABLE 8 Single Crystal Parameters of Form 2 Molecular formulaC₂₃H₃₁Cl₂FN₃O₉P Molecular weight  614.38 Crystal system Monoclinic Spacegroup P2₁ a 12.8315(3) Å, α 90°, b  6.14530(10) Å, β 91.752(2)°, c17.6250(4) Å, γ 90° V 1389.14(5) Å³ Z   2 D_(c) 1.469 g · cm⁻¹ μ 3.196mm⁻¹ Source, λ Cu—K, 1.54178 Å F(000)  640 T 293(2) K Data truncated to0.80 Å θ_(max) 62.23° Completeness 91.1% Reflections 3528 Uniquereflections 2562 R_(int)   0.0227

Example 22-3 Single Crystal X-Ray Crystallography of S_(P)-4 (Form 2)

FIG. 12 depicts an X-ray Crystal Structure (ORTEP—anisotropic) S_(P)-4(Form 2). A crystal structure of the methylene chloride solvate ofS_(P)-4 (Form 2), C₂₃H₃₁N₃PO₉FCl₂, yields a monoclinic space group P2₁(systematic absences 0k0: k=odd) with a=12.8822(14) Å, b=6.1690(7) Å,c=17.733(2) Å, β=92.045(3)°, V=1408.4(3)Å³, Z=2 and d_(calc)=1.449g/cm³. X-ray intensity data were collected on a Rigaku Mercury CCD areadetector employing graphite-monochromated Mo-K_(α) radiation (λ=0.71073Å) at a temperature of 143K. Preliminary indexing was performed from aseries of twelve 0.5° rotation images with exposures of 30 seconds. Atotal of 648 rotation images were collected with a crystal to detectordistance of 35 mm, a 2θswing angle of −12°, rotation widths of 0.5° andexposures of 30 seconds: scan no. 1 was a φ-scan from 315° to 525° atω=10° and χ=20°; scan no. 2 was an ω-scan from −20° to 5° at χ=−90° andφ=315°; scan no. 3 was an ω-scan from −20° to 4° at χ=−90° and φ=135°;scan no. 4 was an ω-scan from −20° to 5° at χ=−90° and φ=225°; scan no.5 was an ω-scan from −20° to 20° at χ=−90° and φ=45°. Rotation imageswere processed using CrystalClear (CrystalClear: Rigaku Corporation,1999), producing a listing of unaveraged F² and σ(F²) values which werethen passed to the CrystalStructure (CrystalStructure: Crystal StructureAnalysis Package, Rigaku Corp. Rigaku/MSC (2002)) program package forfurther processing and structure solution on a Dell Pentium IIIcomputer. A total of 7707 reflections were measured over the ranges5.48≦2θ≦50.04°, −14≦h≦15, −7≦k≦6, −19≦1≦21 yielding 4253 uniquereflections (R_(int)=0.0180). The intensity data were corrected forLorentz and polarization effects and for absorption using REQAB (minimumand maximum transmission 0.824, 1.000).

The structure was solved by direct methods (SIR97, SIR97: Altomare, A.,M. Burla, M. Camalli, G. Cascarano, C. Giacovazzo, A. Guagliardi, A.Moliterni, G. Polidori & R. Spagna (1999). J. Appl. Cryst., 32,115-119). Refinement was by full-matrix least squares based on F² usingSHELXL-97 (SHELXL-97: Sheldrick, G. M. (2008) Acta Cryst., A64,112-122). All reflections were used during refinement. The weightingscheme used was w=1/[σ²(F_(o) ²)+0.0472P²+0.4960P] where P=(F_(o)²+2F)/3. Non-hydrogen atoms were refined anisotropically and hydrogenatoms were refined using a “riding” model. Refinement converged toR₁=0.0328 and wR₂=0.0817 for 4046 reflections for which F>4σ(F) andR₁=0.0348, wR₂=0.0838 and GOF=1.056 for all 4253 unique, non-zeroreflections and 358 variables (R₁=Σ∥F_(o)∥−∥F_(c)∥/Σ|F_(o)|; wR₂={Σw(F_(o) ²−F_(c) ²)²/Σw(F_(o) ²)²}^(1/2); GOF={Σw(F_(o) ²−F_(c)²)²/(n−p)}^(1/2); where n=the number of reflections and p=the number ofparameters refined). The maximum Δ/α in the final cycle of least squareswas 0.000 and the two most prominent peaks in the final differenceFourier were +0.312 and −0.389 e/Å³. The Flack absolute structureparameter refined to −0.06(6) thus corroborating the stereochemistry ofthe title compound.

Table 1 lists cell information, data collection parameters, andrefinement data. Final positional and equivalent isotropic thermalparameters are given in Table 2. Anisotropic thermal parameters are inTable 3. (“ORTEP-II: A Fortran Thermal Ellipsoid Plot Program forCrystal Structure Illustrations”. C. K. Johnson (1976) ORNL-5138.)representation of the molecule with 30% probability thermal ellipsoidsdisplayed.

TABLE 9 Summary of Structure Determination of Compound S_(P)-4•CH₂Cl₂.Formula: C₂₃H₃₁N₃PO₉FCl₂ Formula weight:  614.38 Crystal class:monoclinic Space group: P2₁ (#4) Z   2 Cell constants: a 12.8822(14) Å b6.1690(7) Å c 17.733(2) Å β 92.045(3)° V 1408.4(3) Å³ μ 3.48 cm⁻¹crystal size, mm 0.42 × 0.12 × 0.10 D_(calc) 1.449 g/cm³ F(000)  640Radiation: Mo—K_(α)(λ = 0.71073 Å) 2θ range 5.48-50.04° hkl collected:−14 ≦ h ≦ 15; −7 ≦ k ≦ 6; −19 ≦ 1 ≦ 21 No. reflections measured: 7707No. unique reflections: 4253 (R_(int) = 0.0180) No. observed reflections4046 (F > 4σ) No. reflections used in refinement 4253 No. parameters1358 R indices (F > 4σ) R₁ = 0.0328 wR₂ = 0.0817 R indices (all data R₁= 0.0348 wR₂ = 0.0838 GOF:   1.056 Final Difference Peaks, e/Å³ +0.312,−0.389

Example 22-4 Single Crystal X-Ray Crystallography of S_(P)-4 (Form 3)

FIG. 13 shows an X-ray crystal structure for S_(P)-4 Form 3. There, thisfigure shows a view of molecules of Form 3 from the crystal structureshowing the numbering scheme employed. Anisotropic atomic displacementellipsoids for the non-hydrogen atoms are shown at the 50% probabilitylevel. Hydrogen atoms are displayed with an arbitrarily small radius.

The structure solution was obtained by direct methods, full-matrixleast-squares refinement on F² with weighting w⁻¹=σ²(F_(o)²)+(0.0512P)²+(0.6810P), where P=(F_(o) ²+2F_(c) ²)/3, anisotropicdisplacement parameters, empirical absorption correction using sphericalharmonics, implemented in SCALE3 ABSPACK scaling algorithm. FinalwR²={Σ[w(F_(o) ²−F_(c) ²)²]/Σ[w(F_(o) ²)²]^(1/2)}=0.0796 for all data,conventional R₁=0.0294 on F values of 2486 reflections withF_(o)>4a(F_(o)), S=1.068 for all data and 377 parameters. Final Δ/σ(max)0.001, Δ/σ(mean), 0.000. Final difference map between +0.211 and −0.334e Å⁻³.

TABLE 10 Single Crystal Parameters of Form 3 Molecular formulaC₂₃H₃₀Cl₃F₁N₃O₉P₁ Molecular weight  648.82 Crystal system MonoclinicSpace group P21 a 12.9257(4) Å, α 90°, b  6.18080(10) Å, β 96.399(2)°, c18.0134(4) Å, γ 90° V 1430.15(6) Å³ Z   2 D_(c) 1.507 g · cm⁻¹ μ 3.977mm⁻¹ Source, λ Cu Kα, 1.54178 Å F(000)  672 T 100(1) K Crystal Colorlessneedle, 0.22 × 0.03 × 0.02 mm Data truncated to 0.80 Å θ_(max) 74.41°Completeness 69.1% Reflections 3062 Unique reflections 2607 R_(int)  0.0198

Example 23 Stability at Elevated Temperatures and Relative Humidity

A sample of R_(P)-4 was stored in a humidity chamber at 40° C. and 75%relative humidity for one week, and the sample was reanalyzed by XRPD.The powder pattern obtained for R_(P)-4 showed no substantial changeduring the course of the experiment, meaning that no change in solidform was observed. This should be contrasted to a sample of 4, whichdeliquesced within about 16 hours upon storage at 40° C. and 75%relative humidity. Indeed, an illustration of the deliquescent nature of4 is illustrated by the following. A sample of 4 was passed through a250 μm sieve then samples were stored at 40° C./75% RH and 25° C./53%relative humidity and visual observations were taken at regularintervals. The results are given in Table 4.

TABLE 11 Stability of 4 to elevated relative humidity. Conditions t =1.5 h t = 4.5 h t = 6.5 h t = 8.5 h t = 73 h 40° C./ Deliquescence — — —— 75% RH 25° C./ No Sticky Partial Almost Deliquescence 53% RHdeliquescence solid deliquescence complete deliquescence

Upon storage at 40° C. and 75% relative humidity a sample of S_(P)-4deliquesced inside 16 hours. For instance, a sample of S_(P)-4 wasground with a pestle and mortar, and then successively passed through500 and 250 μm sieves to yield the sample as a fine powder. Samples ofthis material were stored at 40° C. and 75% relative humidity and 25° C.and 53% RH and visual observations were taken at regular intervals. Theresults are given in Table 5.

TABLE 12 Stability of S_(P)-4 to elevated relative humidity. Conditionst = 1.5 h t = 4.5 h t = 104 h 40° C./75% RH No deliquescenceDeliquescence — 25° C./53% RH No deliquescence No deliquescence Nodeliquescence

XRPD analysis of the sample after storage at 25° C. and 53% RH for 104hours showed no significant changes in the diffractograms producedindicating that no form change had occurred.

Example 24 Fourier Transform—Infrared (FT-IR) Spectrometry

Data were collected on a Perkin-Elmer Spectrum One fitted with auniversal Attenuated Total Reflectance (ATR) sampling accessory. Thedata were collected and analyzed using Spectrum v5.0.1 software.

The IR spectrum obtained for 4, R_(P)-4, and S_(P)-4 are shown in FIGS.5-7, respectively. Selected peaks, in wavenumbers (cm⁻¹) are recitedbelow:

4: ˜1680, ˜1454, ˜1376, ˜1205, ˜1092, ˜1023 (FIG. 14);

R_(P)-4: ˜1742, ˜1713, ˜1679, ˜1460, ˜1377, ˜1259, ˜1157, ˜1079 (FIG.15); and

S_(P)-4 (Form 1): ˜1743, ˜1713, ˜1688, ˜1454, ˜1378, ˜1208, ˜1082 (FIG.16).

Example 25 Differential Scanning Calorimetry (DSC) Thermo-GravimetricAnalysis (TGA)

DSC data were collected on a TA Instruments Q2000 equipped with a 50position auto-sampler. The calibration for thermal capacity was carriedout using sapphire and the calibration for energy and temperature wascarried out using certified indium.

Modulated temperature DSC was carried out on typically 0.8-1.2 mg ofeach sample, in a pin-holed aluminum pan, using an underlying heatingrate of 2° C.min⁻¹ and temperature modulation parameters of ±0.2°C.min⁻¹ and 40 seconds. A purge of dry nitrogen at 50 ml.min⁻¹ wasmaintained over the sample.

The instrument control software was Advantage for Q Series v2.8.0.392and Thermal Advantage v4.8.3 and the data were analyzed using UniversalAnalysis v4.3A.

DSC data were collected on a Mettler DSC 823e equipped with a 34position auto-sampler. The instrument was calibrated for energy andtemperature using certified indium. Typically 0.8-1.2 mg of each sample,in a pin-holed aluminum pan, was heated at 10° C.min⁻¹ from 25° C. to250° C. A nitrogen purge at 50 ml.min⁻¹ was maintained over the sample.The instrument control and data analysis software was STARe v9.20.

TGA data were collected on a Mettler TGA/SDTA 851e equipped with a 34position auto-sampler. The instrument was temperature calibrated usingcertified indium. Typically 8-12 mg of each sample was loaded onto apre-weighed aluminum crucible and was heated at 10° C.min⁻¹ from ambienttemperature to 350° C. A nitrogen purge at 50 ml.min⁻¹ was maintainedover the sample. The instrument control and data analysis software wasSTARe v9.20.

DSC analysis of 4 showed a single broad endotherm with an onset of 58.7°C. (ΔH 14J.g⁻¹) confirmed to be due to molecular relaxation during theglass transition by further modulated DSC analysis (FIG. 17). TGAanalysis of 4 showed no weight loss before decomposition above 240° C.,confirming the material to be non-solvated. As the XRPD analysis of 4confirmed the material to be amorphous, modulated DSC analysis wasundertaken in an attempt to calculate the glass transition temperature,which was found to be 57° C.

DSC analysis showed a single sharp endotherm with an onset of 136.2° C.(ΔH 76 J.g⁻¹) confirmed to be a melt by hot stage microscopy. See FIG.18. TGA analysis of R_(P)-4 showed no weight loss before decompositionabove 240° C., confirming the material to be non-solvated.

DSC analysis of S_(P)-4 showed a single broad endotherm with an onset of93.9° C. (ΔH 43 J.g⁻¹) confirmed to a melt by hot stage microscopy. SeeFIG. 19. TGA analysis of S_(P)-4 showed no weight loss beforedecomposition above 240° C., confirming the material to be non-solvated.

Example 26 Gravimetric Vapour Sorption (GVS) SMS DVS Intrinsic

Sorption isotherms were obtained using a SMS DVS Intrinsic moisturesorption analyzer, controlled by SMS Analysis Suite software. The sampletemperature was maintained at 25° C. by the instrument controls. Thehumidity was controlled by mixing streams of dry and wet nitrogen, witha total flow rate of 200 ml.min⁻¹. The relative humidity was measured bya calibrated Rotronic probe (dynamic range of 1.0-100% RH), located nearthe sample. The weight change, (mass relaxation) of the sample as afunction of % RH was constantly monitored by the microbalance (accuracy±0.005 mg).

Typically 5-20 mg of sample was placed in a tared mesh stainless steelbasket under ambient conditions. The sample was loaded and unloaded at40% RH and 25° C. (typical room conditions). A moisture sorptionisotherm was performed as outlined below (2 scans giving 1 completecycle). The standard isotherm was performed at 25° C. at 10% RHintervals over a 0.5-90% RH range.

TABLE 13 Method Parameters for SMS DVS Intrinsic Experiments ParametersValues Adsorption - Scan 1 40-90 Desorption/Adsorption - Scan 2 90-0,0-40 Intervals (% RH) 10 Number of Scans 2 Flow rate (ml · min⁻¹) 200Temperature (° C.) 25 Stability (° C. min⁻¹) 0.2 Sorption Time (hours) 6hour time out

The sample was recovered after completion of the isotherm andre-analyzed by XRPD.

GVS analysis showed R_(P)-4 to be non-hygroscopic exhibiting reversibleuptake of approximately 0.2 wt % of water from 0 to 90% relativehumidity. Re-analysis of the sample by XRPD after the GVS experimentshowed no change in form.

A sample of S_(P)-4 was ground with a pestle and mortar, and thensuccessively passed through 500 and 250 μm sieves to yield the sample asa fine powder that was then analyzed using a modified single cyclemethod. The sample was taken from 40% RH (approximately ambient) to 60%RH, instead of 90% for the standard method, and then cycled to 0% andback to 40% RH. This analysis showed S_(P)-4 to be non-hygroscopic up to60% RH, with reversible uptake of ˜0.2% by weight of water from 0 to 60%RH.

Example 27 Thermodynamic Aqueous Solubility

Aqueous solubility was determined by suspending a sufficient amount ofcompound in water to give a maximum final concentration of ≧10 mg.ml⁻¹of the parent free-form of the compound. The suspension was equilibratedat 25° C. for 24 hours then the pH was measured. The suspension was thenfiltered through a glass fiber C filter into a 96 well plate. Thefiltrate was then diluted by a factor of 101. Quantitation was by HPLCwith reference to a standard solution of approximately 0.1 mg.ml⁻¹ inDMSO. Different volumes of the standard, diluted and undiluted samplesolutions were injected. The solubility was calculated using the peakareas determined by integration of the peak found at the same retentiontime as the principal peak in the standard injection.

TABLE 14 HPLC Method Parameters for Solubility Measurements Type ofmethod: Reverse phase with gradient elution Column: Phenomenex Luna, C18(2) 5 μm 50 × 4.6 mm Column Temperature 25 (° C.): Standard Injections(μl): 1, 2, 3, 5, 7, 10 Test Injections (μl): 1, 2, 3, 10, 20, 50Detection: 260, 80 Wavelength, Bandwidth (nm): Flow Rate (ml · min⁻¹): 2Phase A: 0.1% TFA in water Phase B: 0.085% TFA in acetonitrile Time(min) % Phase A % Phase B Timetable: 0.0 95  5 1.0 80 20 2.3  5 95 3.3 5 95 3.5 95  5 4.4 95  5

Analysis was performed under the above-noted conditions on an AgilentHP1100 series system equipped with a diode array detector and usingChemStation software vB.02.01-SR1.

TABLE 15 Aqueous solubility result for R_(P)-4, 4, and S_(P)-4. pH ofUnfiltered Sample ID mixture Solubility/mg · ml⁻¹ Comments R_(P)-4 7.121.58 Suspension 4 7.03 6.11 Residual solid S_(P)-4 6.88 5.65 Residualsolid

Example 28 Chemical Purity Determination by HPLC

Various HPLC conditions can be used to determine the chemical purity ofthe compounds disclosed herein. One such example is disclosed above inrelation to the thermodynamic aqueous solubility studies. Anotherexample is disclosed below.

HPLC Conditions:

LC: Waters Alliance 2695 Separations Module, Waters 2996 PDA detectorand Waters Empower 2 Software (Version 6.00) Column: Phenomenex LunaC18(2); 4.6 × 50 mm; 3 μm Flow rate: 1.2 mL/min Injection Volume: 10 μLMobile phase: Solvent A: 95% Water with 5% Methanol and 10 mM AmmoniumAcetate; pH ~5.3 Solvent B: MeOH with 10 mM Ammonium Acetate Gradient:hold at 0% B       3 min 0-47% B     3-4 min hold at 47% B     4-10 min47%-74% B    10-11 min hold at 74% B   11-13.5 min return to 0% B13.5-13.6 min hold at 0% B 13.6-15.5 min

Under these conditions, the purity of 4, R_(P)-4, and S_(P)-4 wasdetermined to be ˜99.6, ˜99%, and ˜99.5%, respectively. It is noted thathigher purities can be realized by optimizing the methods disclosedabove.

Inspection of the XRPD diffractograms shows that the two crystallinesingle diastereoisomers gave clearly different XRPD patterns.Additionally, there was a clear difference in the melting point of thetwo crystalline diastereoisomers, with R_(P)-4 having a considerablyhigher onset than S_(P)-4 (136° C. vs. 94° C.).

Example 29 Additional Separation Methods

The following SFC separation (conditions listed below) yielded adequateseparation of a mixture of the diastereomers, R_(P)-4 and S_(P)-4.

Preparative Method: Analytical Method: Chiralpak AS-H (2 × 25 cm) SN#07-8656 Chiralpak AS-H (25 × 0.46 cm) 20% methanol/CO₂ (100 bar) 20%methanol/CO₂ (100 bar) 50 ml/min, 220 nm. 3 ml/min, 220 nm. Conc.: 260mg/30 ml methanol, inj vol.: 1.5 ml

The following SFC separation (conditions listed below) yielded adequateseparation of a mixture of the diastereomers, R_(P)-4 and S_(P)-4.

Preparative Method: Analytical Method: Chiralpak IA(2 × 15 cm) 802091Chiralpak IA (15 × 0.46 cm) 30% isopropanol(0.1% DEA)/CO₂, 100 bar 40%methanol(DEA)/CO₂, 100 bar 60 mL/min, 220 nm. 3 mL/min, 220 nm. injvol.: 2 mL, 20 mg/mL methanol

TABLE 16 Summary of results from the batch characterization of R_(P)-4,4, and S_(P)-4. Analysis R_(P)-4 4 S_(P)-4 Proton NMR Singlediastereoisomer 1:1 Mixture of Single diastereoisomer diastereoisomersXRPD Crystalline - different Amorphous Crystalline - different DSC fromS_(P)-4 Endotherm; 59° C. from R_(P)-4 Endotherm; melt - 136° C.Endotherm; melt - 94° C. TGA No wt loss, No wt loss, decomposition No wtloss, decomposition >240° C. >240° C. decomposition >240° C. IR Seeabove See above See above Aq Solubility 1.58 6.11 5.65 (mg · ml⁻¹) HPLCPurity 96.9% 99.6% 99.5% 40° C./75% RH No form change Deliquescenceinside 1.5 h Deliquescence inside 4.5 h 25° C./53% RH — Deliquescence Noform change GVS Non-hygroscopic up to 90% — Non-hygroscopic up to 60% RHRH

Example 30 X-Ray Crystallography of 8 (S_(P)-Isomer)

Compound 8 (S_(P)-isomer), C₁₈H₂₁N₂PO₇, crystallizes in the monoclinicspace group P2₁ (systematic absences 0k0: k=odd) with a=5.3312(4)Å,b=15.3388(8)Å, c=23.7807(13)Å, β=92.891(3)°, V=1942.2(2)Å³, Z=4, andd_(calc)=1.397 g/cm³. X-ray intensity data were collected on a BrukerAPEXII CCD area detector employing graphite-monochromated Mo-Kαradiation (λ=0.71073 Å) at a temperature of 100(1)K. FIGS. 20A and 20Bshow molecules numbered 1 and 2, respectively, of the asymmetric unit.

Preliminary indexing was performed from a series of thirty-six 0.5°rotation frames with exposures of 30 seconds. A total of 3608 frameswere collected with a crystal to detector distance of 70.00 mm, rotationwidths of 0.5° and exposures of 20 seconds:

scan type 2θ ω φ χ frames φ −35.50 279.40 27.32 48.96 725 φ 24.50 22.3135.56 69.08 692 ω −13.00 321.68 247.79 69.08 95 φ 34.50 204.08 28.21−92.80 293 φ −30.50 310.60 214.10 54.21 361 φ 32.00 304.67 24.47 50.72722 φ −35.50 122.14 316.59 −78.84 720

Rotation frames were integrated using SAINT (Bruker (2009) SAINT. BrukerAXS Inc., Madison, Wis., USA.) producing a listing of unaveraged F² andσ(F²) values which were then passed to the SHELXTL (Bruker (2009)SHELXTL. Bruker AXS Inc., Madison, Wis., USA.) program package forfurther processing and structure solution on a Dell Pentium 4 computer.A total of 6909 reflections were measured over the ranges 1.58≦θ≦25.09°,−6≦h≦6, −18≦k≦18, −28≦1≦28 yielding 6909 unique reflections(Rint=0.0581). The intensity data were corrected for Lorentz andpolarization effects and for absorption using SADABS (Sheldrick, G. M.(2007) SADABS. University of Gottingen, Germany.) (minimum and maximumtransmission 0.6093, 0.7452).

The structure was solved by direct methods (SHELXS-97 (Sheldrick, G. M.(2008) Acta Cryst. A64, 112-122.)). Refinement was by full-matrix leastsquares based on F² using SHELXL-97 (Sheldrick, G. M. (2008) Acta Cryst.A64, 112-122.). All reflections were used during refinement. Theweighting scheme used was w=1/[σ²(F_(o) ²)+(0.0000P)²+14.0738P] whereP=(F_(o) ²+2F_(c) ²)/3. Non-hydrogen atoms were refined anisotropicallyand hydrogen atoms were refined using a riding model. Refinementconverged to R1=0.0847 and wR2=0.1899 for 6173 observed reflections forwhich F>4σ(F) and R1=0.0963 and wR2=0.1963 and GOF=1.119 for all 6909unique, non-zero reflections and 512 variables(R1=Σ∥F_(o)|−|F_(c)∥/Σ|F_(o)|; wR2=[Σw(F_(o) ²−F_(c) ²)²/Σw(F_(o)²)²]^(1/2); GOF=[Σw(F_(o) ²−F_(c) ²)²/(n−p)]^(1/2); where n=the numberof reflections and p=the number of parameters refined). The maximum Δ/σin the final cycle of least squares was 0.000 and the two most prominentpeaks in the final difference Fourier were +0.402 and −0.559 e/Å³.

TABLE 17 Summary of Structure Determination of Compound 8 (S_(P)-isomer)Empirical formula C₁₈H₂₁N₂PO₇ Formula weight  408.34 Temperature 100(1)K Wavelength 0.71073 Å Crystal system monoclinic Space group P2₁ Cellconstants: a 5.3312(4) Å b 15.3388(8) Å c 23.7807(13) Å β 92.891(3)°Volume 1942.2(2) Å³ Z   4 Density (calculated) 1.397 Mg/m³ Absorptioncoefficient 0.185 mm⁻¹ F(000)  856 Crystal size 0.40 × 0.10 × 0.08 mm³Theta range for data collection 1.58 to 25.09° Index ranges −6 ≦ h ≦ 6,−18 ≦ k ≦ 18, −28 ≦ 1 ≦ 28 Reflections collected 6909 Independentreflections 6909 [R(int) = 0.0581] Completeness to theta = 25.09° 99.6%Absorption correction Semi-empirical from equivalents Max. and min.transmission 0.7452 and 0.6093 Refinement method Full-matrixleast-squares on F² Data/restraints/parameters 6909/1/512Goodness-of-fit on F²   1.119 Final R indices [I > 2sigma(I)] R1 =0.0847, wR2 = 0.1899 R indices (all data) R1 = 0.0963, wR2 = 0.1963Absolute structure parameter 0.1(2) Largest diff. peak and hole 0.402and −0.559 e · Å⁻³

Example 31 Biological Activity

Replicon containing cells were seeded at either 3,000 cells/well (50 μL)in 96-well white/opaque plates, or 1,500 cells/well (25 μL) in 384-wellwhite/opaque plates. 50 μL of 2× compound were added in the 96 wellplate or 25 μL of 2× compound were added in the 384 well plate. Theplates were incubated at 37° C. in a humidified 5% CO₂ atmosphere for 4days. After incubation, Bright-Glo reagent (50 μL for 96-well plate, or25 μL for 384-well plate) was added to measure the firefly luciferasereporter for HCV replication. Percent inhibition was calculated againstthe no drug control.

Compound HCV Replicon Activity (μM) 4 0.58 R_(P)-4 2.87 S_(P)-4 0.13

R_(P)-4 and S_(P)-4 have been demonstrated to have broad genotypecoverage. For example, both have been shown to be active againsthepatitis C virus, genotypes 1-4.

The subject matter of U.S. patent application Ser. No. 12/053,015 andU.S. Provisional Patent Application Nos. 61/179,923, filed May 20, 2009,and 61/319,513, filed Mar. 31, 2010, are hereby incorporated byreference in their entireties. The subject matter of all citedreferences is hereby incorporated by reference. In the event that themeaning of an incorporated term conflicts with the meaning of a termdefined herein, the meaning of the terms contained in the presentdisclosure control over the meaning of the incorporated terms.

1.-81. (canceled)
 82. A process for preparing (R_(P)-4):

which comprises reacting an isopropyl-alanyl-phosphoramidate (C′) with3′-O-protected or unprotected 2′-deoxy-2′-fluoro-2′-C-methyluridine (3′)and a basic reagent to obtain protected or unprotected (R_(P)-4′):

wherein Z is a protecting group or hydrogen, and LG′ is a leaving group.83. The process of claim 82, wherein Z is hydrogen.
 84. The process ofclaim 82, wherein the leaving group is an aryloxide substituted with atleast one electron withdrawing group.
 85. The process of claim 82,wherein the leaving group is p-nitrophenoxide, 2,4-dinitrophenoxide, orpentafluorophenoxide.
 86. The process of claim 82, wherein nochromatography is performed.
 87. A process for preparing (S_(P)-4):

which comprises reacting an isopropyl-alanyl-phosphoramidate (C) with3′-O-protected or unprotected 2′-deoxy-2′-fluoro-2′-C-methyluridine (3′)and a basic reagent to obtain protected or unprotected (S_(P)-4′):

wherein Z is a protecting group or hydrogen, and LG′ is a leaving group.88. The process of claim 87, wherein Z is hydrogen.
 89. The process ofclaim 87, wherein the leaving group is an aryloxide substituted with atleast one electron withdrawing group.
 90. The process of claim 87,wherein the leaving group is p-nitrophenoxide, 2,4-dinitrophenoxide, orpentafluorophenoxide.
 91. The process of claim 87, wherein nochromatography is performed.